Redox regulation by SOD2 modulates colorectal cancer tumorigenesis through AMPK‐mediated energy metabolism

Colorectal cancer (CRC) is a common malignancy. Many reports have implicated aberrant mitochondrial activity in the progression of CRC, with particular emphasis on the dysregulation of redox signaling and oxidative stress. In this study, we focused on manganese superoxide dismutase (MnSOD/SOD2), a key antioxidant enzyme, which maintains intracellular redox homeostasis. Current literature presents conflicting mechanisms for how SOD2 influences tumorigenesis and tumor progression. Here, we explored the role of SOD2 in CRC specifically. We found high levels of SOD2 expression in CRC tissues. We carried out a series of experiments to determine whether knockdown of SOD2 expression in CRC cell lines would reverse features of tumorigenesis. We found that reduced SOD2 expression decreased cell proliferation, migration, and invasion activity in CRC cells. Results from an additional series of experiments on mitochondrial function implicated a dual role for SOD2 in promoting CRC progression. First, proper level of SOD2 helped CRC cells maintain mitochondrial function by disposal of superoxide (O2.−). Second, over‐expression of SOD2 induced H2O2‐mediated tumorigenesis by upregulating AMPK and glycolysis. Our results indicate that SOD2 may promote the occurrence and development of CRC by regulating the energy metabolism mediated by AMPK signaling pathways.


| INTRODUCTION
Colorectal cancer (CRC) is one of the most common cancers in the world. Unlike many other diseases in recent years, CRC has shown a trend of increasing morbidity and mortality in younger individuals. 1 Due to the lack of specific symptoms and implementation of effective screening program, CRC screening rates remain relatively low in the general population. 2 Moreover, the long-term survival rate of advanced CRC patients is still poor. 3 The most well-known role of mitochondria in the cell is regulation of cellular energy metabolism; however, these organelles also play a vital role in differentiation, 4 signal transduction, and apoptosis 5 in eukaryotic cells. Energy metabolism in most cells consists of both mitochondrial oxidative phosphorylation and glycolysis. However, cancer cells predominantly rely on glycolysis for adenosine triphosphate (ATP), a phenomenon known as the Warburg effect. 6 Reactive oxygen species (ROS) are a by-product of mitochondrial oxidative phosphorylation that seem to also act as signaling molecules for both cell proliferation and cell death. 7 Abbreviations: ECAR, extracellular acidification rate; OCR, oxygen consumption rate; shRNA, short hairpin RNA.
The alteration of ROS levels has thus been associated with various processes involved in the occurrence and development of cancer, including aging, 8 carcinogenesis, 9 tumor progression, 10 metastasis, 11 and tumor survival. 12 Under normal physiological conditions, the cells have a comprehensive antioxidant defense system for eliminating excessive ROS and regulating redox status. SOD2, a significant player in the antioxidant defense system, governs ROS homeostasis by converting superoxide (O 2 .− ) into hydrogen peroxide (H 2 O 2 ). SOD2 has attracted much attention in recent years due to its potential role in the diagnosis, treatment, and prognosis of various cancers. [13][14][15] Some reports have classified SOD2 as a tumor suppressor due to its lower expression in some cancers. [16][17][18] For example, the combination of SOD2 and cisplatin significantly inhibited ovarian tumor growth by inducing apoptosis. 19 In hepatoma cells, inhibition of SOD2 promoted tumorigenesis and produced a more malignant phenotype with metabolic transformation via a reduction β-catenin and hypoxia-inducible factor-1α. 20 Serum expression of hepatocarcinogenesis markers was enhanced in mice deficient for hepatocyte-specific SOD2. 20 However, results from other studies seem to be at odds with the concept of SOD2 being a tumor suppressor. For example, SOD2 showed upregulation in advanced tumors, specifically. 21,22 In the absence of SOD2 expression, tumor cell proliferation, migration, and metastasis were inhibited in ovarian clear cell carcinoma, accompanied by a significant decrease of Akt phosphorylation. 23 Likewise, overexpression of SOD2 promoted the growth and plating efficiency of esophageal squamous cell carcinoma; 24 SOD2 also enhanced the invasion ability of gastric mucosal cancer cells via increasing intracellular reduced thiol levels. 25 Although increased expression of SOD2 in CRC has been shown by immunohistochemical assessment previously, 26 its role in the occurrence and development of CRC has been studied very little. In this study, we explored SOD2 expression in CRC tissues and the effects of changes in SOD2 expression on CRC cell proliferation and migration. We further investigated the underlying mechanisms by which SOD2 mediates tumorigenesis in CRC.

| Immunohistochemistry
SOD2 expression from 54 cancer patients and monocarboxylate transporter (MCT4) expression from 24 cancer patients was quantified using immunohistochemistry. Paraffin-embedded 4.5-µm-thick tissue sections were stained for mouse monoclonal antibody obtained from Santa Cruz Biotechnology (SOD2 mouse monoclonal sc-133254, MCT4 rabbit polyclonal sc-50329). Deparaffinization of the sections was performed using a solution of dimethyl benzene, and rehydration was performed using a graded alcohol series.
Endogenous catalase was blocked using hydrogen peroxide. To restore the antigen, tissue sections were treated three times with a 10 mM citrate buffer heated in a microwave for 5 minutes each.
Following the citrate treatment, the sections were incubated with mouse/rabbit monoclonal antibody (dilution 1:100 in 1× TBST) overnight, and processed with a secondary antibody amplification system for 20 minutes. After counterstaining with DAB (PV-9000, Beijing) and hematoxylin, the sections were dehydrated and sealed with neutral balsam for examination and storage. Using Image-Pro

| Cell culture
F I G U R E 2 Knockdown of SOD2 expression decreased tumorigenicity of CRC cells. A, SOD2 expression profiles in selected CRC cell lines. B, SOD2 knockdown in SW480 and DLD-1 cells. C, Clone formation assays showed decreased proliferation in shSOD2 CRC cell lines compared with control cells. D, Migration ability of controls and shSOD2 CRC cells were analyzed by transwell assay. E, The migration capacity of controls and shSOD2 CRC cells were evaluated using a wound-healing assay. F, The invasion ability of controls and shSOD2 CRC cells were evaluated using a Transwell and Matrigel assays. Control cells were designed to express the plko.1 vector. Statistical analysis was performed using Student's t test. Data are presented as the means ± standard deviation (n

| Clonogenicity assay
The cells were seeded into a six-well plate at a concentration of 2000 cells/well and 500 cells/well, respectively, and cultured for about 14 days with 5% CO 2 at 37℃. Clonogenicity was analyzed after staining the colonies with crystal violet. Images were obtained using the microscope's built-in camera (Nikon ECLIPSE TS100).

| Wound-healing assay
The cells cultured in six-well plates were incubated overnight, yielding confluent monolayers for a wound-healing assay. Wounds were simulated using a pipette tip scratch and photographs were taken immediately after the scratch using a microscope camera (Nikon ECLIPSE TS100) at 24, 48, and 72 hours, respectively. The distance of migration by the cell monolayer towards closing the wounded area during each time period was measured and quantified by Image J v 2.4.1.7 (National Institutes of Health, Bethesda, MD).
The final concentration of relative reagents used in the cell culture F I G U R E 3 Mitochondrial function in shSOD2 CRC cells. A, Mitochondrial function was measured by extracellular flow analysis using the Seahorse Biosciences XF analyzer. Cell number was used to adjust all measurements of OCR. B, Mitochondrial membrane potential was determined by TMRM and normalized by nuclear stain (Hoechst) fluorescence reading. C, Total cellular ATP content was measured in shSOD2 CRC cells and control cells with a luciferase-based ATP detection kit and normalized by protein content. D, Mitochondrial ATP content was measured in shSOD2 CRC cells and control cells with a luciferase-based ATP detection kit and normalized by protein content. Control cells were designed to express the plko.1 vector. Statistical analysis was performed using Student's t test. All quantitative data represent the mean ± SE of three independent experiments. ATP, adenosine triphosphate; CRC, colorectal cancer; OCR, oxygen consumption rate; SOD2, superoxide dismutase. **P < .01, ***P < .001 [Color figure can be viewed at wileyonlinelibrary.com] medium were as follows: CAT: 800 KU/L; H 2 O 2 : 200/300 μM; compound C: 4 μM.

| Transwell migration assay and matrigel invasion assay
The matrigel invasion assay was carried out using the Transwell migration assay with matrigel. The configuration of media in the Transwell was as follows: serum-free DMEM in the Transwell itself,  F I G U R E 4 Glycolytic activity of shSOD2 CRC cells. A, Glycolysis capacity was measured by extracellular flow analysis using Seahorse Biosciences XF analyzer. Cell number was used to adjust all measurements of OCR yielding quantitative results for glycolytic capacity. B, Glucose uptake was analyzed in shSOD2 CRC cells and control cells using a 2-DG uptake assay, and was then normalized by protein content. C, To quantify lactate levels, the cells were cultured for 48 hours in complete medium and extracellular lactate level was measured in the media and normal cells. D, The MCT4 protein content in shSOD2 CRC cells and control cells was analyzed by Western blot. Control cells were designed to express the plko.1 vector. Statistical analysis was performed using Student's t test. All quantitative data represent the mean ± SE of three independent experiments. CRC, colorectal cancer; OCR, oxygen consumption rate; SOD2, superoxide dismutase. **P < .01, ***P < .001 [Color figure can be viewed at wileyonlinelibrary.com] ZHOU ET AL.

| ATP measurements
ATP measurements were done as described previously. 27

| Measurement of mitochondrial superoxide
Superoxide anion radical levels in mitochondria were detected using Mito SOX. 28

| Statistical analysis
Statistical analysis was performed by the GraphPad Prism Software 6 using either Student's t test or paired Student's t test. Data are presented as the means ± SEM. P < .05 was considered significant, whereas P < .01 and P < .001 were considered highly significant.
All experiments were performed in triplicate and were performed independently at least three times.

| CRC tissues showed higher SOD2 expression compared with normal tissues
To determine the expression of SOD2 in CRC, we first analyzed SOD2 protein levels in 54 paraffinized tissue samples obtained from clinical cases of CRC from a local hospital in southern China.
Immunohistochemistry of tissue sections revealed higher expression of SOD2 in cancer tissues compared with histologically normal tissue adjacent to the tumor (Figure 1).

| SOD2 knockdown decreases tumorigenicity of CRC cells
We then examined the expression of SOD2 in a series of CRC cell lines ( Figure 2A). To investigate the potential role of SOD2 in the tumorigenesis of CRC, we chose two CRC cell lines with relatively low (SW480) and high (DLD-1) levels of SOD2, respectively (Figure 2A). We first generated stable SOD2 knockdown cell lines by introducing SOD2specific shRNA to both SW480 and DLD-1 cells. Empty expression vectors were used as controls. The SOD2 knockdown effects were confirmed by Western blot analysis ( Figure 2B). We found that with decreased SOD2 expression, colony formation decreased markedly in both SW480 and DLD-1 cells ( Figure 2C). Similarly, the cell migration ability (Figures 2D and 2E) and invasion capacity ( Figure 2F) determined by Transwell and wound-healing assays all declined significantly in SW480 and DLD-1 cells with knockdown of SOD2 expression.

| SOD2 knockdown decreased mitochondrial function
As SOD2 localizes to mitochondria, we next investigated whether modulation of tumorigenesis by SOD2 could be mediated by alterations in mitochondrial function. We first measured respiration using the Seahorse XF96 Extracellular Flux Analyzer, and found that basal, ATP-coupled, and maximal respiration decreased with the knockdown of SOD2 ( Figure 3A). In addition, knockdown of SOD2 expression in CRC cells also decreased MMP ( Figure 3B), as well as total ATP content ( Figure 3C) and mitochondrial ATP content ( Figure 3D).

| SOD2-dependent regulation of glycolytic activity in CRC cells
Malignant cells predominantly rely on glycolysis for ATP. To further study the potential role of SOD2 expression in energy metabolism, we measured glycolytic activity, glucose uptake, and other features of glycolysis in SOD2 knockdown cells. ECAR was decreased in SOD2 knockdown cells ( Figure 4A). Glucose uptake decreased ( Figure 4B), and the glycolysis markers L-lactate ( Figure 4C) and MCT4 ( Figure 4D) also declined significantly in SOD2 knockdown cells.

| SOD2 regulates oxidative stress to promote the migration of CRC cells via AMPK phosphorylation
To explore the potential molecular pathway connecting SOD2 modulation with tumorigenesis and energy metabolism, we investigated the ROS balance and its effect on SOD2 expression in CRC cells. We evaluated the oxidative stress using superoxide anion levels in the cells. Interestingly, the mitochondrial superoxide anion levels increased as detected by MitoSox ( Figure 5A), while hydrogen peroxide levels as detected by catalase activity assays decreased ( Figure 5B)  All the quantitative data represent the mean ± SE of three independent experiments. *P < .05, **P < .01, ***P < .001, ****P < .0001. AMPK, AMP-activated protein kinaseα; CRC, colorectal cancer; SOD2, superoxide dismutase cells. We found that the reduction of SOD2 significantly decreased the levels of phosphorylated AMPK ( Figure 6A). H 2 O 2 treatment also enhanced the AMPK phosphorylation in CRC cells (Figures 6B and 6C).

| SOD2 regulation of tumorigenesis and glycolysis is mediated by AMPK activation
We then explored the role of AMPK in SOD2-mediated regulation of tumorigenesis and energy metabolism. We found that the inhibition of AMPK by compound C significantly decreased cell migration in each of two different cell lines with different SOD2 expression levels ( Figure 7A). Inhibition of AMPK also reduced glycolytic activity as measured by L-lactate levels in the medium ( Figure 7B). Direct measurement of glycolysis using seahorse XF96 Extracellular Flux Analyzer again showed decreased glycolysis, glycolytic capacity, and glycolytic reserve in cells cultured with compound C ( Figure 7C).
To confirm the connection between regulation of glycolysis with modulation of SOD2 in CRC cells, we analyzed CRC tissue samples for levels of MCT4, a marker of glycolysis in cancer cells. 30,31 In these clinical samples, MCT4 levels were higher in CRC tissues compared with adjacent normal tissues ( Figure 8A,B cancers displaying increased SOD2 levels. 32 We observed that levels of SOD2 are high in CRC tissues obtained from patients in a local Chinese population. In addition, we showed that migration and invasion ability of CRC cells was attenuated in SOD2 knockdown cells.
These results are consistent with the idea that a more malignant phenotype is associated with upregulation of SOD2. Indeed, SOD2 levels are reported to increase during metastatic progression and in highly aggressive tumor cell lines. [33][34][35][36] Prior studies have also shown that elevated antioxidant enzyme expression is a necessary survival adaptation during tumor progression. 37 This suggests SOD2 has an oncogenic role via alleviation of oxidative stress in cancers.

| Regulation of energy metabolism in CRC
Oxidative stress and mitochondrial dysfunction are typical characteristics in a variety of malignant tumors. SOD2 regulates redox homeostasis by converting superoxide into hydrogen peroxide. In addition, hydrogen peroxide activates multiple intracellular pathways. Our studies showed that upregulation of SOD2 maintains mitochondrial function, indicated by reduction in mitochondrial ATP production and MMP in SOD2 knockdown CRC cells. Such protection through maintaining mitochondrial integrity was previously reported in other cancer types. 38 Enhanced glycolysis is also a common characteristic in the majority of malignant tumors. Our study provided a link between enhanced SOD2 expression and increased glycolytic activity. It is interesting to note that decreased glycolytic activity due to loss of SOD2 was previously reported in breast cancer. 29 Taking all these results together, it seems that SOD2 modulates energy metabolism during development and progression of CRC.

| AMPK-mediated, SOD2-dependent tumorigenesis in CRC
AMPK, as a cellular metabolic master switch, has been implicated in most cancers and found to regulate growth, 39 migration, 40 autophagy, 41 and apoptosis. 42 High AMPK levels were also observed in CRC stem cells, which served to promote the survival of CRC cells. 43 In our experiments, AMPK phosphorylation levels were reduced in SOD2 knockdown CRC cells, while addition of exogenous H 2 O 2 activated AMPK. AMPK inhibition decreased the migration ability of CRC cells, accompanied with a reduction in glycolysis. These results indicate that SOD2 promotes tumorigenesis via AMPK-dependent pathways in CRC.
In line with our study, other authors also found that AMPK inhibition attenuated aerobic glycolysis and inhibited malignant phenotype in pancreatic cancer. 44 Overall, our studies demonstrated elevated SOD2 expression and glycolysis in CRC cells, elucidating a mechanistic link between SOD2-mediated control of redox status and AMPK activation with subsequent effects on energy metabolism. These results may provide a new perspective for the future development of CRC therapeutics.