J Cancer Prev 2021; 26(1): 41-53
Published online March 30, 2021
© Korean Society of Cancer Prevention
1Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, 2Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, 3Department of Pathology, Hallym University Dongtan Sacred Heart Hospital, Hwaseong, Korea, 4Laboratory of Immunology, Division of Biotechnology Review and Research-III, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Colon tumors develop more frequently in male than in female. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays differential roles in the stage of tumorigenesis. The purpose of this study was to investigate the role of Nrf2 on colitis-associated tumorigenesis using Nrf2 knockout (KO) female mice. Azoxymethane (AOM) and dextran sulfate sodium (DSS)-treated wild-type (WT) and Nrf2 KO female mice were sacrificed at week 2 and 16 after AOM injection. Severity of colitis, tumor incidence, and levels of inflammatory mediators were evaluated in AOM/DSS-treated WT and Nrf2 KO mice. Furthermore, qRT-PCR, Western blot abnalysis, and ELISA were performed in colon tissues. At week 2, AOM/DSS-induced colon tissue damages were significantly greater in Nrf2 KO than in WT mice. At week 16, tumor numbers (> 2 mm size) were significantly lower in both the proximal and distal colon in Nrf2 KO compared to WT. The overall incidences of adenoma/cancer of the proximal colon and submucosal invasive cancer of the distal colon were reduced by Nrf2 KO. The mRNA and protein expression levels of NF-κB-related mediators (i.e., iNOS and COX-2) and Nrf2-related antioxidants (i.e., heme oxygenase-1 and glutamate-cysteine ligase catalytic subunit) were significantly lower in the Nrf2 KO than in WT mice. Interestingly, the protein level of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) was higher in AOM/DSS-treated Nrf2 KO than in WT mice. Our results support the oncogenic effect of Nrf2 in the later stage of carcinogenesis and upregulation of tumor suppressor 15-PGDH might contribute to the repression of colitis-associated tumorigenesis in Nrf2 KO female mice.
Keywords: Colitis-associated carcinogenesis, Nrf2 knockout, AOM/DSS mouse model, Colon cancer, 15-PGDH
Colorectal cancer (CRC) is the third leading cause of cancer-associated death in both male and female in the United States, with an estimation of 149,500 new cancer cases and 52,980 cancer deaths, in 2021 . In South Korea, male have a higher incidence rate of age-adjusted CRC than female . However, elderly female over 65 years of age show a higher mortality of CRC and a lower 5-year survival rate compared to their age-adjusted male . CRC shows sex differences worldwide in the incidence and the developing site (proximal or distal colon), and underlying mechanisms .
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays a pivotal role in adaptive cellular defense response to a variety of stimuli such as oxidation, proteotoxic stress, metabolic stress, and inflammation [4,5]. In normal cells under non-stress conditions, Nrf2 levels and activity are kept relatively low by its rapid proteasomal degradation which is facilitated by Kelch-like ECH-associated protein 1 (Keap1) . Under stress conditions, however, Nrf2 dissociates from Keap1 and then translocates to the nucleus . Nrf2 activates anti-oxidant enzymes, such as heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidoreductase 1 (NQO1), glutamate-cysteine ligase catalytic subunit (GCLC), and glutamate-cysteine ligase modifier subunit (GCLM), that maintain cellular homeostasis and exhibit anti-inflammatory and anti-tumor activity [7,8]. The anti-inflammatory mechanisms of Nrf2 include suppression of pro-inflammatory cytokines such as interleukin (IL)-6, IL-1β, and TNF-α by blocking the NF-κB signaling [9,10].
Recently, there have been many reports on the dual roles of Nrf2 in carcinogenesis. Activation of Nrf2 in normal cells prevents them from transformation into cancer cells, whereas activation of Nrf2 in transformed or cancerous cells promotes their growth and survival . Meta-analysis showed that patients with high Nrf2 expression had a lower overall survival rate and disease-free survival compared to those with low Nrf2 expression . In addition, high Nrf2 levels were associated with poor prognosis in CRC patients . Furthermore, overexpression of Nrf2 target genes such as HO-1 and NQO1 has been reported in prostate cancer and CRC, respectively [14,15]. Further, Nrf2 overexpression accounts for chemotherapy resistance in many malignancies including gastric and colon cancer [13,16].
Azoxymethane (AOM) and dextran sulfate sodium (DSS) treatments [17-19] are the most widely used protocols for the establishment of animal models of colon carcinogenesis [20-22]. This model well reflects multistep nature of tumor development and progression based on the aberrant crypt foci (ACF)-adenoma-carcinoma sequence with the relevant molecular alterations . Therefore, the Nrf2 KO (Nrf2−/−) AOM/DSS mouse model could be a useful tool to clarify the role of Nrf2 by sex in colitis and colon tumorigenesis.
We previously reported that 17β-estradiol (10 mg/kg) reduced the expression of Nrf2 upregulated in AOM/DSS-treated male ICR mice and suppressed the occurrence of colitis-related CRC to a level similar to that of females . However, the inhibitory effect of 17β-estradiol (10 mg/kg) on expression of Nrf2 in C57BL/6 background male mice (wild-type, WT) was not sufficient to inhibit tumorigenesis in the distal colon . In contrast, in the absence of Nrf2 (Nrf2 KO), 17β-estradiol (10 mg/kg) strongly inhibited tumorigenesis in the distal colon through an Nrf2-independent estrogen receptor beta (ERβ)-related signaling pathway . From these observations, we hypothesized that the contribution of Nrf2 to colitis-associated colon tumorigenesis could vary by sex. To test this hypothesis, we investigated the effect of Nrf2 on colitis-associated colon tumorigenesis in AOM/DSS-treated Nrf2 KO (Nrf2−/−) and WT (Nrf2+/+) female mice.
Heterozygous Nrf2 KO (Nrf2+/−) mice of C57BL6/129SV background generated by the laboratory of Yuet Wai Kan  were kindly provided by Prof. Y.-J. Surh of Seoul National University. WT (Nrf2+/+) and homozygous Nrf2 KO (Nrf2−/−) mice were obtained by crossing the Nrf2 heterozygous (Nrf2+/−) mice (Fig. 1A) as described previously . The mice were housed in cages at 23°C with a 12/12-hour light/dark cycle under specific pathogen-free conditions. Genomic DNA (gDNA) obtained using DNeasy® Blood & Tissue Kit (Qiagen Gmbh, Hilden, Germany) was used as the template DNA (listed in Table 1) and the PCR products were visualized under a ChemiDocTM Imaging System (Bio-Rad Laboratories, Inc., Munich, Germany) (Fig. 1B). All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Seoul National University Bundang Hospital (BA1705-223/043-01) and performed in accordance with the ARRIVE (Animals Research: Reporting In Vivo Experiments) protocol.
For the induction of colitis-associated CRC, 2.5% (w/v) DSS (Cat no 160110; MP Biomedicals, Solon, OH, USA) was supplied in the drinking water for 7 days, one week following the injection of AOM (10 mg/kg) (Cat no A5486; Sigma-Aldrich, St. Louis, MO, USA) counted as day 0 [24,26]. WT and Nrf2 KO female mice were randomized into the following groups. Group 1: WT control mice (n = 11), Group 2: AOM/DSS-treated WT mice (n = 9-12), Group 3: Nrf2 KO control mice (n = 10), Group 4: AOM/DSS-treated Nrf2 KO mice (n = 13-16). The animals were euthanized by CO2 asphyxiation at week 2 (10 weeks of age) and 16 (24 weeks of age) after AOM injection (Fig. 1C).
Clinical symptoms were evaluated using the disease activity index (DAI), which includes loss of body weight, stool characterization, and hematochezia [27,28]. The DAI was scored by two researchers in a blinded manner as described elsewhere .
The colons were opened longitudinally, and stool was washed out with PBS. The colon length was measured from the cecum to the rectum using a ruler. Polypoid lesions with a diameter ≤ 2 mm or > 2 mm were independently counted by two researchers in a blinded manner [27,28] as described previously . The tumor incidence was determined as the percentage of mice bearing more than one tumor.
The extracted colon was divided into proximal and distal portions. The proximal colon up to 1.5 cm from the ileocecal valve, the rectum up to 1.5 cm from the anal verge, and colonic segments containing any gross polyps were fixed with phosphate-buffered formalin and embedded in paraffin. The sections (5 mm) were stained with hematoxylin and eosin (H&E). The classification of adenoma and adenocarcinoma, and the specification of the depth of adenocarcinoma invasion into the colonic tissues as mucosal or submucosal invasion were performed in a blinded manner . The histological severity was assessed using a microscopic damage score reflecting colonic epithelial damage and depth of inflammatory cell infiltration .
The levels of myeloperoxidase (MPO), IL-6, and IL-1β in the colonic tissues were measured using a mouse MPO ELISA kit (Cat no HK210; Hycult Biotechnology, Uden, The Netherlands), a mouse IL-6 Quantikine ELISA kit (Cat no M6000B; R&D Systems Inc., Minneapolis, MN, USA), and a mouse IL-1β/IL-1F2 Quantikine ELISA kit (Cat no MLB00C; R&D Systems Inc.), respectively, according to the manufacturer’s instructions. All assays were performed in triplicate.
Total RNA was extracted from the colon tissues using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA). For qRT-PCR, 2 µg of total RNA was reverse transcribed using High Capacity cDNA Reverse Transcription kit according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). The cDNA was used to perform qRT-PCR using speciﬁc primers (listed in Table 1) in a ViiA7 instrument (Applied Biosystems). The expression levels were normalized to that of
Colon tissue was lysed with RIPA buffer (Cell Signaling Technology, Beverly, MA, USA) containing protease and phosphatase inhibitors. Total protein was separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Western blot analysis was performed with specific primary antibodies (listed in Table 2). The signals were then detected with an enhanced chemiluminescence (ECL) kit (GE Healthcare Biosciences, Buckinghamshire, UK). The band intensity was quantified by densitometric analysis using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
All statistical analyses were conducted using GraphPad Prism, version 5.01 (GraphPad Software, Inc., San Diego, CA, USA) and PASW Statistics for Windows, version 18.0 (IBM Corp., Armonk, NY, USA). Data are expressed as the mean ± SEM. Statistical significance was examined with the Mann–Whitney test or Fisher’s exact test.
To investigate the potential role of Nrf2 in AOM/DSS-induced inflammation and tumorigenesis in female mice, a total of four experimental groups were prepared as presented in Figure 1C. Two WT groups comprised female control mice (Group 1) and AOM/DSS-treated female mice (Group 2). In Nrf2 KO groups, two groups also comprised female control mice (Group 3) and AOM/DSS-treated female mice (Group 4).
First, we measured the colitis-associated symptoms including DAI, colonic epithelial damage, and colon length shortening. The DAI was calculated by summing the body weight change, stool consistency, and rectal bleeding, and by dividing the result by three. In the AOM/DSS-treated animal model, body weight loss is a sensitive indicator of colitis severity , which is linked to colon tumorigenesis. The body weight loss caused by AOM/DSS treatment was observed in both WT and Nrf2 KO groups without the effect of Nrf2 KO at week 2 (Fig. 2A and 2B). However, at week 16, significant body weight loss was observed in both Nrf2 KO control and AOM/DSS groups (Fig. 2A and 2C,
To determine the effect of Nrf2 on colitis-associated tumorigenesis in females, we counted the tumor number by macroscopic assessment, assessing both the size and the location, at week 16. Tumors were well developed at week 16 in both the proximal and distal regions of colon in AOM/DSS-treated WT and Nrf2 KO mice (Fig. 3A). Interestingly, tumor numbers, especially those > 2 mm in size, were significantly lower in Nrf2 KO mice than in WT mice both in proximal and distal regions of colon (Fig. 3A,
To further assess the effect of Nrf2 KO on inflammatory mediators at the molecular level, NF-κB-mediated expression of pro-inflammatory enzymes and cytokines, and their gene levels were measured in colon tissue at week 2. The mRNA expression levels of inducible nitric oxide synthase (
We further measured mRNA expression of the Nrf2-mediated anti-oxidant enzymes at week 2. The mRNA expression of
At week 16, the mRNA expression levels of
We further measured mRNA and protein expression of the Nrf2 target genes encoding anti-oxidant enzymes at week 16. The mRNA expression of
To figure out the molecular signatures on Nrf2-associated tumorigenesis in female mice, we analyzed the colonic expression of ERβ and 15-hydroxy prostaglandin dehydrogenase (15-PGDH). There was no change in the protein expression of ERβ by AOM/DSS treatment in WT mice, but the expression of ERβ was significantly decreased by AOM/DSS treatment in the Nrf2 KO group (Fig. 5F,
Our results showed that AOM/DSS-mediated colonic epithelial damages were significantly worse in Nrf2 KO female mice than in WT at the colitis stage (week 2). However, at the tumorigenesis stage (week 16), the number of tumors exceeding 2 mm in size was significantly lower in both the proximal and distal colons in Nrf2 KO compared to WT female mice. Furthermore, the overall adenoma/cancer incidence of the proximal colon and submucosal invasive cancer of the distal colon was decreased in Nrf2 KO mice than in WT animals. Interestingly, the mRNA or protein expression of NF-κB-related mediators and Nrf2-related antioxidant enzymes was significantly lower in Nrf2 KO mice, while the protein expression level of 15-PGDH was higher in Nrf2 KO than in WT mice. These results suggest that Nrf2 acts differentially, in a way that it exerts an anti-inflammatory effect in the colitis stage and a carcinogenic effect in the tumorigenesis stage.
Nrf2 is a key modulator of the adaptive response to a variety of environmental and endogenous stresses [32-36]. Recently, there are several reports that Nrf2 contributes to weight gain. The body weight of adult Nrf2 KO mice was lower compared to WT mice fed a normal diet . Nrf2 was involved in weight gain in male mice during space travel by maintaining homeostasis of white adipose tissue . Decreased expression of peroxisome proliferator-activated receptor γ due to Nrf2 deficiency prevented weight gain and obesity from a high fat diet and environmental stress caused by space travel [37,38]. In our previous study, the body weight of male control mice was not affected by Nrf2 KO at week 16 . In the present study, however, the body weight of the Nrf2 KO female control or AOM/DSS-treated group was significantly lower compared to that of the WT group at week 16. Several studies have shown that Nrf2 KO mice are more susceptible to inflammatory diseases [39,40]. Khor et al. reported that mRNA and protein expression of COX-2 and iNOS induced by AOM/DSS treatment increased in colon tissue of Nrf2 KO male mice compared to WT animals . A study using peritoneal macrophages prepared from WT male and Nrf2 KO male mice showed that iNOS protein expression was more strongly inhibited by phenethyl isothiocyanate and curcumin treatment in WT than in Nrf2 KO mice . At week 2, colon length shortening and colonic epithelial damages were also affected similarly to that of Nrf2 KO-mediated weight loss. In males, the colon length of the control group and colonic epithelial damages induced by AOM/DSS were not affected by Nrf2 KO . However, females were easily affected by Nrf2 KO, as revealed by shortening of the colon length and increased colon epithelial damage. At the molecular level, the protein expression of the proinflammatory mediator iNOS induced by AOM/DSS treatment was lower in WT females than in WT males ; however, sex differences in iNOS expression disappeared by Nrf2 KO. These results indicate that the contribution of Nrf2 to homeostasis maintenance and inflammatory defense mechanisms is greater in female mice than in males. It is speculated that female mice are more susceptible to Nrf2 KO than males.
Recently, Nrf2 was known to be involved in maintaining cancer cell growth and invasion by metabolic reprogramming, inhibiting cancer cell apoptosis, and enhancing the ability of cancer stem cells to self-renew in the tumorigenesis stage. More importantly, aberrant activation of Nrf2 was associated with chemoresistance of cancer cells and poor prognosis . In the previous study, we also observed that Nrf2 and Nrf2-related antioxidant genes were highly expressed in AOM/DSS-treated male and female mice, which accelerated cancer development . Therefore, we expected that tumorigenicity would be weaker in the Nrf2 KO group than in WT mice. As expected, the incidence of tumors was lower in the Nrf2 KO female mice than in the WT females, especially those with tumor sizes greater than 2 mm. In particular, only the incidence of submucosal invasive cancer at the distal colon was reduced by Nrf2 KO in females. In the previous study, the tumor incidence in male mice was not affected by Nrf2 KO, so there was no difference from the tumor incidence in WT mice . Interestingly, the anti-tumorigenic effect of 17β-estradiol was stronger in the AOM/DSS-treated Nrf2 KO group than in WT male mice .
Several studies have demonstrated the tumor suppressor function of 15-PGDH, an enzyme that converts prostaglandin E2 into an inactive metabolite [43-45]. The expression of 15-PGDH was strongly reduced in various human malignancies such as colon cancer, lung cancer, and gastric cancer compared to normal tissues [46,47]. 15-PGDH is abundantly expressed in the normal colonic mucosa, and loss of 15-PGDH is associated with CRC development [46,48]. In the present study, there was no change in the protein expression of 15-PGDH by AOM/DSS treatment compared to the female control in the 16-week WT group, but the expression of 15-PGDH was strongly increased in the AOM/DSS-treated Nrf2 KO female group compared to its control. In the male group, the expression of 15-PGDH was strongly inhibited in both WT and Nrf2 KO mice by AOM/DSS treatment, and there was no change by Nrf2 KO (data not shown). Interestingly, there was no change in the expression of 15-PGDH and ERβ by AOM/DSS treatment in the WT female mice, but it was observed that protein expression of 15-PGDH and ERβ was oppositely regulated in the Nrf2 KO female group. Taken together our results regarding ERβ and 15-PGDH in the Nrf2 KO mice suggest that 15-PGDH might contribute to the suppression of colitis-associated tumorigenesis in Nrf2 KO female mice. However, further experiments are needed to clarify this supposition.
In conclusion, our study shows the possibility that Nrf2 KO suppresses the colitis-associated tumorigenesis by upregulating tumor suppressor 15-PGDH in females. The elevated Nrf2 levels are associated with the development of aggressive CRC, and 15-PGDH may be a useful therapeutic target depending on sex in CRC patients.
We are grateful to Prof. Young-Joon Surh (Seoul National University College of Pharmacy, Seoul, Korea) for providing heterozygous Nrf2 knockout (Nrf2+/−) mice. This research was supported by a grant from the National Research Foundation of Korea (NRF) funded by the government of the Republic of Korea (2019R1A2C2085149).
No potential conflicts of interest were disclosed.
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