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. 2010 Aug 1;10(3):223-31.
doi: 10.4161/cbt.10.3.12207. Epub 2010 Aug 3.

NADPH oxidase 4 is an oncoprotein localized to mitochondria

Kelly A Graham  1 , Mariola Kulawiec, Kjerstin M Owens, Xiurong Li, Mohamed Mokhtar Desouki, Dhyan Chandra, Keshav K Singh
Affiliations

NADPH oxidase 4 is an oncoprotein localized to mitochondria

Kelly A Graham et al. Cancer Biol Ther. .

Abstract

Reactive oxygen species (ROS) are known to be involved in many physiological and pathological processes. Initially ROS-producing NADPH oxidase (NOX) proteins were thought to be present in phagocytes. However, recent studies have demonstrated that NOX proteins are expressed in many other cell types and tissues. NOX family members' expression and function seems to vary from tissue to tissue. We determined the expression of the NOX family of proteins (NOX1-5) in normal breast tissue and breast tumors. Our study revealed that normal breast tissues express NOX1, 4 and 5 genes. Similar pattern of expression was revealed in a breast epithelial cell line. We found that NOX4 was overexpressed in the majority of breast cancer cell lines and primary breast tumors. NOX4 was also overexpressed in ovarian tumors. Overexpression of NOX4 in normal breast epithelial cells resulted in cellular senescence, resistance to apoptosis, and tumorigenic transformation. Overexpression of NOX4 in already transformed breast tumor cells also showed increased tumorigenicity. Strong evidence suggests that regulation of these processes occurs through NOX4 generation of ROS in the mitochondria. We demonstrate that the NOX4 protein contains a 73 amino acid long mitochondrial localization signal at the N-terminus that is capable of transporting a passenger protein GFP into the mitochondria. Treatment of NOX4 overexpressing cells with catalase resulted in decreased tumorigenic characteristics. Together, this study provides evidence for an oncogenic function for NOX4 protein localized to mitochondria and suggests that NOX4 is a novel source of ROS produced in the mitochondria. This study also identifies a possible treatment of NOX4-induced breast cancer by antioxidant treatment.

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Figures

Figure 1
Figure 1
(A) NOX expression in normal breast cell line and normal breast tissue. RT-PCR was used to analyze the gene expression of the NADPH family members in normal breast tissue (I) and the MCF12A normal breast cell line (II). (B) NOX4 is overexpressed in malignant breast cell lines and breast tumors. RT-PCR was conducted to determine expression of the NOX4 gene in cell lines and breast tissue and tumor samples. The NOX4 gene is overexpressed in most breast cancer cell lines and breast tumors (T) when compared to normal breast tissue (N). (C) NOX4 is overexpressed in breast tumors. IHC analysis was done on tissue array (TARP5) containing breast carcinomas of different grades. (I) Representative positive breast carcinoma case. (II) Representative negative breast carcinoma case. (III) Graph representing NOX4 expression in all breast tumors assayed. (IV) Graph representing NOX4 expression in breast tumors stratified by grade.
Figure 2
Figure 2
NOX4 is overexpressed in ovarian tumors. IHC analysis was performed on tissue array (TARP5) containing ovarian carcinomas of different grades. (A) Representative positive ovarian carcinoma case. (B) Representative negative ovarian carcinoma case. (C) Graph representing NOX4 expression in all ovarian tumors assayed. (D) Graph representing NOX4 expression in ovarian tumors stratified by grade.
Figure 3
Figure 3
NOX4 localizes to the mitochondria. pEGFP-N2-NOX4MLS construct was used to transfect NIH3T3 cells. (A) MitoTracker staining determined mitochondrial location, DAPI staining determined nuclear location, GFP staining determined location of the NOX4 protein. (B) Western blot analyses of pEGFP-N2-NOX4MLS expressing cells showing NOX4 import into the mitochondria.
Figure 4
Figure 4
Generation of an MCF12A cell line with stable overexpression of NOX4. A(I) RT-PCR analysis demonstrates a higher expression of the NOX4 gene when compared to the empty vector control. A(II) Western blot analysis was performed using the membrane fraction isolated from wild-type and NOX4 overexpressing MCF12A cells. Amido Black was used as a loading control. (B) Measurement of NOX4 activity using a nitroblue terazolum (NBT) assay. NBT reduction was normalized to cell number. (C) DHE and DCF assays were conducted to analyze the amount of ROS produced by NOX4 overexpressing MCF12A cells when compared to the empty vector (Mock) control. (D) Beta-galactosidase assays were conducted to determine cellular senescence in MCF12A (I) and MDA-MB435 (II) cell lines.
Figure 5
Figure 5
NOX4 overexpression induces resistance to cell death. Apoptosis induced by intrinsic (TRAIL) and extrinsic (Etoposide) factors was measured by quantification of Annexin staining. Values represent fold differences in apoptosis normalized to the untreated control.
Figure 6
Figure 6
Tumorigenic transformation of breast epithelial cells overexpressing NOX4. Parallel analyses were conducted in MCF12A and MDA-MB435 cells overexpressing NOX4. (A) Anchorage-independent cell growth was assayed. Colonies represents an average value for assays repeated in triplicate with analysis of six fields of view per assay. (B) Invasion was assayed using a matrigel boyden chamber. Matrigel invasion assays for MCF12A cells were completed in duplicate with three chambers per assay. Matrigel invasion assays for MDA-MB435 cells were completed once with three chambers per assay. For each analysis, 10 fields of view were captured and the number of cells per field of view was quantified. Mean number of cells were determined by taking the average of the ten fields of view. The average of the two analyses were calculated and plotted as mean cells/field. (C) Cell doubling time was analyzed for NOX4 and Mock MCF12A and MDA-MB435 cell lines. Cell doubling time was calculated as described in the methods. (D) Catalase treatment inhibits Matrigel invasion induced by NOX4 overexpression. Matrigel invasion assays were completed once for the MCF12A cells and twice for the MDA-MB435 cells with three chambers per cell type per assay. Invasion was quantified as outlined in (B). Stars denote statistically significant differences between treated and untreated NOX4 overexpressing cell lines.
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