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.