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

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.

Prognostic stratification of high-risk gastrointestinal stromal tumors in the era of targeted therapy.

BACKGROUND: Recently, a trial of adjuvant imatinib for primary R0-resected intermediate and high-risk gastrointestinal stromal tumors (GISTs) significantly improved recurrence-free survival. But identifying patients having higher chances of recurrence will reduce economic losses and prevent adverse side effects caused by adjuvant treatment. METHODS: Tissue samples from 93 patients with high-risk GISTs were studied for p16, CD34, and CD44 protein expression and mutations of KIT and PDGFRA gene. Clinicopathologic, immunohistochemical, and mutation results were compared with clinical outcome by univariate and multivariate analyses. RESULTS: KIT mutations were observed in 75 cases (81%) including 46 exon 11 deletion mutations and 31 deletions affecting codons 557-558. A novel 12 bp deletion mutation (KHNG484-488) on KIT exon 9 was detected in a small intestinal GIST. For recurrence-free survival, R0 resection, organ-confined disease stage, and female sex are better prognostic factors in univariate analysis and disease stage was the only factor predicting recurrence (P = 0.02) in multivariate analysis. In overall survival, mutation types, presence of mutation, location of GISTs, and mitosis were significant by univariate analysis. After multivariate analysis, mitotic counts and presence of KIT mutation corresponded to independent prognostic factors. Moreover, mitosis, KIT exon 11 deletion mutation, and deletions affecting exon 557-558 predict recurrence in R0-resected high-risk GISTs (P < 0.05). CONCLUSION: Prognostic stratification in high-risk GISTs will help identify patients with high-risk GIST who may benefit from adjuvant therapy.