To facilitate functional investigation of the role of
NADPH oxidase 1 (NOX1) and associated
reactive oxygen species in
cancer cell signaling, we report herein the development and characterization of a novel mouse
monoclonal antibody that specifically recognizes the C-terminal region of the
NOX1 protein. The antibody was validated in stable NOX1 overexpression and knockout systems, and demonstrates wide applicability for Western blot analysis, confocal microscopy, flow cytometry, and immunohistochemistry. We employed our NOX1 antibody to characterize NOX1 expression in a panel of 30 human
colorectal cancer cell lines, and correlated
protein expression with NOX1
mRNA expression and
superoxide production in a subset of these cells. Although a significant correlation between oncogenic RAS status and NOX1
mRNA levels could not be demonstrated in
colon cancer cell lines, RAS mutational status did correlate with NOX1 expression in human
colon cancer surgical specimens. Immunohistochemical analysis of a comprehensive set of tissue microarrays comprising over 1,200
formalin-fixed,
paraffin-embedded tissue cores from human epithelial
tumors and inflammatory disease confirmed that NOX1 is overexpressed in human colon and small intestinal
adenocarcinomas, as well as
adenomatous polyps, compared to adjacent, uninvolved intestinal mucosae. In contradistinction to prior studies, we did not find evidence of NOX1 overexpression at the
protein level in
tumors versus histologically normal tissues in prostate, lung, ovarian, or
breast carcinomas. This study constitutes the most comprehensive histopathological characterization of NOX1 to date in cellular models of
colon cancer and in normal and malignant human tissues using a thoroughly evaluated
monoclonal antibody. It also further establishes NOX1 as a clinically relevant therapeutic target in colorectal and small
intestinal cancer.