Panoply™ Human ADAM17 Knockdown Stable Cell Line

ADAM17 is believed to promote tumor development by facilitating cell proliferation and migration. Here, researchers explored the role of ADAM17 and its effect on integrin pathway activation in regulating the malignant characteristics of hepatocellular carcinoma (HCC) cells and tissues. Using human tissue microarrays and a diethylnitrosamine-induced HCC mouse model, ADAM17 was found to be positively correlated with active integrin β1. ADAM17 and active integrin β1 levels were elevated in HCC tissues compared to adjacent liver tissues, and active integrin β1 levels were correlated with tumor size and TNM staging. High levels of ADAM17 and active integrin β1 in tumor tissue were significantly associated with poor prognosis in HCC patients. RNAi-mediated ADAM17 knockdown and integrin β1 blockade significantly inhibited HCC cell migration and invasion, while ADAM17 overexpression produced the opposite effect. In an orthotopic xenograft model, ADAM17 interference attenuated intrahepatic growth and metastasis of HCC cells. Levels of active integrin β1, p-FAK, p-AKT, MMP-2, and MMP-9 were decreased in ADAM17 knockdown cells. ADAM17 knockdown significantly inhibited the translocation of the Notch1 intracellular domain to the nucleus, while overexpression of the Notch1 intracellular domain restored translocation and enhanced integrin β1 activation. These data suggest that ADAM17, by regulating integrin β1 activation and the Notch1 signaling pathway, is a crucial determinant of malignant characteristics. Inhibiting ADAM17 may offer a viable therapeutic potential for preventing HCC metastasis.

To assess the effect of ADAM17 on intracellular signaling in HCC cells, active integrin β1 in ADAM17-knockdown SMMC-7721 cells and ADAM17-overexpressing SMMC-7721 cells was analyzed by flow cytometry. As shown in Figure 1A, ADAM17 knockdown reduced the level of active integrin β1, while ADAM17 overexpression increased the level of active integrin β1. The FAK/Akt pathway is an important downstream component of integrin signaling and also promotes the activation of Rac and Rho. Immunofluorescence analysis of phosphorylated AKT pSer473 showed increased levels of ADAM17-mediated AKT activation (Figure 1B). Similar to the immunofluorescence results, Western blot data showed that phosphorylation of AKT and FAK (pTyr397), as well as the expression of MMP-2 and MMP-9, were inhibited in ADAM17 knockdown cells (Figure 1C). To investigate the role of Notch in ADAM17-mediated integrin β1 activation, researchers transfected lentiviruses carrying the Notch1 intracellular domain-pcw107 or pcw107 (empty vector) into ADAM17 knockdown SMMC-7721 cells. As shown in Figures 1D and E, ADAM17 knockdown reduced the level of the Notch-1 intracellular domain in the nucleus, while transfection of the active form of Notch-1 (Notch-1 intracellular domain, NIC) into ADAM17 knockdown HCC cells significantly activated integrin β1, but did not affect integrin expression. Overexpression of NIC also enhanced the migration and invasion capabilities of ADAM17 knockdown cells, and this enhancement was inhibited by an integrin β1 blocking antibody (Figure 1F). These results indicate that ADAM17 plays a crucial role in mediating the extracellular-intracellular signaling activation of integrin β1 through the Notch-1 signaling pathway.

Figure 1. ADAM17 is involved in regulation of integrin β1 signaling via nuclear translocation of the Notch-1 intracellular domain. (Li Y, et al., 2018)