NADPH Oxidase 1 Activity and ROS Generation Are Regulated by Grb2/Cbl-Mediated Proteasomal Degradation of NoxO1 in Colon Cancer Cells

Abstract

The generation of reactive oxygen species (ROS) is required for proper cell signaling, but must be tightly regulated to minimize deleterious oxidizing effects. Activation of the NADPH oxidases (Nox) triggers ROS production and, thus, regulatory mechanisms exist to properly control Nox activity. In this study, we report a novel mechanism in which Nox1 activity is regulated through the proteasomal degradation of Nox organizer 1 (NoxO1). We found that through the interaction between NoxO1 and growth receptor-bound protein 2 (Grb2), the Casitas B-lineage lymphoma (Cbl) E3 ligase was recruited, leading to decreased NoxO1 stability and a subsequent reduction in ROS generation upon epidermal growth factor (EGF) stimulation. Additionally, we show that EGF-mediated phosphorylation of NoxO1 induced its release from Grb2 and facilitated its association with Nox activator 1 (NoxA1) to stimulate ROS production. Consistently, overexpression of Grb2 resulted in decreased Nox1 activity, whereas knockdown of Grb2 led to increased Nox1 activity in response to EGF. CRISPR/Cas9-mediated NoxO1 knockout in human colon cancer cells abrogated anchorage-independent growth on soft agar and tumor-forming ability in athymic nude mice. Moreover, the expression and stability of NoxO1 were significantly increased in human colon cancer tissues compared with normal colon. Taken together, these results support a model whereby Nox1 activity and ROS generation are regulated by Grb2/Cbl-mediated proteolysis of NoxO1 in response to EGF, providing new insight into the processes by which excessive ROS production may promote oncogenic signaling to drive colorectal tumorigenesis.

Genome-Wide Transcriptional Analysis Reveals the Protection against Hypoxia-Induced Oxidative Injury in the Intestine of Tibetans via the Inhibition of GRB2/EGFR/PTPN11 Pathways

“…As reported in previous studies, GRB2 was related with formation of reactive and oxidative products [55, 56]. ROS are directly involved in gastrointestinal injury.…”

“…Furthermore, the three genes were closed associated with the ROS production. The generation of ROS is tightly regulated by GRB2 in colorectal tumorigenesis [56]. ROS production will be beneficial to EGFR activation [66].…”

NOX1-derived ROS drive the expression of Lipocalin-2 in colonic epithelial cells in inflammatory conditions

“…Interestingly, it has been demonstrated that NOX1 activity could be regulated by proteasomal degradation of NOXO1 in colon cancer cells. 48 In conclusion, our study revealed that production of ROS by NOX1 drives the expression of the bacteriostatic protein, LCN-2, in colonic epithelial cells co-stimulated with TNF-α and IL-17, and that the underlying mechanisms involve a p38MAPK-JNK/ NOXO1/IkBζ axis (Fig. 10).…”

Grb2 carboxyl-terminal SH3 domain can bivalently associate with two ligands, in an SH3 dependent manner

“…Similarly, the constitutive association of Grb2 SH3C with Cbl, Gab2b (lacks the polyproline helix) and LAT has also been reported in the T cell, where Grb2 SH3C interacts with the atypical PXXXR motif of Gab2b 53, 54 and the proline rich region of Cbl, simultaneously. More recently, a trimeric complex involving NoxO1-Grb2-Cbl has been reported where the Grb2 SH3C domain binds NoxO1 peptide “PPVVPTRP”, in addition to Cbl 55 . We surmise that the bivalent mechanism of interaction may be common to several other Grb2 mediated complexes of the immune system, given the abundance of Grb2 ligands that lack the polyproline helix, but has remained elusive, partly due to its transient nature.…”