Inflammation is known to predispose patients to cancer initiation and development (1). For instance, inflammatory diseases such as chronic inflammatory bowel disease, chronic hepatitis, and Helicobacter pylori-induced gastritis have been associated with an increased risk of cancer (2). Tumor initiation occurs as a result of genetic mutations induced by extrinsic mutagens or inherent DNA copying errors. Inflammatory responses lead to the accumulation of mutations even in the absence of extrinsic mutagens (3). For example, activated macrophages and neutrophils produce reactive oxygen species (ROS) and reactive nitrogen species, which induce mutations in normal tissues (1). Therefore, an inadequately resolved chronic inflammation can increase the risk of cancer.

A programmed death 1 (PD-1) is an immune checkpoint expressed on activated T cells, and its engagement with programmed death-ligand 1 (PD-L1) attenuates T cell activation (4). PD-L1 expression in tumors or infiltrating immune cells plays a role in suppressing T cell activation, thereby dampening T cell cytotoxicity and further anti-tumor immune responses (5, 6). Indeed, immune checkpoint inhibitors blocking the PD-1/PD-L1 interaction have shown remarkable results in cancer therapy (7). Most recently, the therapeutic effect of trastuzumab deluxtecan (DS-8201), which is an antibody–drug conjugate, was reported in patients with CRC (8). It showed strong and durable anti-tumor activity in patients with HER2-positive metastatic CRC following two or more previous therapies (8). Furthermore, PD-L1 is expressed in various normal tissues and maintains immune homeostasis (9). For instance, PD-L1-deficient mice show an exacerbated inflammatory response in the colon, suggesting a role for PD-L1 in immune tolerance (10). Studies have revealed that induction of PD-L1 expression can occur in response to inflammatory cytokines such as interferon-gamma (IFN-γ), interleukin-17 (IL-17), and tumor necrosis factor-alpha (TNF-α) (11). Among downstream signaling pathways of inflammation, nuclear factor-κB (NF-κB) has emerged as a key regulator of PD-L1 expression (12). Therefore, chronic inflammation is closely linked to the mechanism of tumor immune evasion (12).

It has been reported that estrogen suppresses intestinal inflammation in clinical and experimental studies [reviewed in (13)], and further colitis-associated cancer (CAC) development in an azoxymethane (AOM)/dextran sodium sulfate (DSS) mouse model (14, 15). Conversely, several studies have revealed that estrogen facilitates tumorigenesis by promoting immunosuppression (e.g. increasing PD-L1 expression on tumor cells) in the tumor microenvironment (TME) (16–18). However, in these studies, the effects of estrogen were evaluated on existing tumor cells. Estrogen receptors (ERs) are classified as ERα and ERβ, both of which have distinct roles: that ERα is oncogenic and promotes cellular proliferation, whereas ERβ is predominantly protective, being anti-proliferative, anti-inflammatory, and anti-tumorigenic (19). In the previous study, 17β-estradiol (E2) treatment induced the expression of ERβ but not ERα in CCD841CoN cells, a female human colonic epithelial cell line. E2 treatment significantly elevated the expression of anti-oxidant enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H-quinone oxidoreductase-1 (NQO1) (20). Furthermore, E2 supplementation decreased protein and mRNA levels of NF-kB-related pro-inflammatory enzymes such as nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) induced by AOM/DSS treatment in the colitis stage (14). These results suggest that estrogen exerts its anti-inflammatory and anti-oxidant effects via ERβ (20).

Nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor maintains redox homeostasis during oxidative stress and suppresses inflammation (21). Consistent with its role, Nrf2 deficiency exacerbates inflammation in various murine models (21). Accordingly, it has been established that Nrf2 plays a role in suppressing the inflammatory response, thus protecting cells from inflammation-induced carcinogenesis (21). Furthermore, we have previously revealed that Nrf2 can mediate the anti-inflammatory effect of E2 in mouse embryonic fibroblast cells (22). This led us to postulate that the effect of E2 would be impaired in Nrf2-deficient mice. However, our recent study showed that the anti-tumorigenic effect of E2 was more prominent in Nrf2 knockout (KO) male mice than in wild-type (WT) male mice (23). These results indicate that Nrf2 activation may not be beneficial under certain conditions. Moreover, Nrf2 activation protects not only normal cells but also cancer cells from oxidative stress which promotes cancer progression (24). More importantly, a recent study has reported that Nrf2 transcriptionally regulates PD-L1 expression (25). In tumor-bearing mice, Nrf2 deficiency decreases the tumor burden and inhibits the function and survival of myeloid-derived suppressor cells (MDSCs) (26), which express high levels of PD-L1 and contribute to the immunosuppressive TME (27).

Based on this background, we hypothesized that estrogen might exert anti-tumorigenic effects and cooperate with Nrf2 deficiency in the immune microenvironment Therefore, this study aimed to determine the sex difference in PD-L1 expression and its correlation with Nrf2 expression in the colon tumor microenvironment of the AOM/DSS-induced CRC mouse model. Also, as part of the sex difference, we tried to confirm the effect of E2 on PD-L1 expression in male mice.

Previously, we reported that E2 more effectively suppressed AOM/DSS-induced tumorigenesis in Nrf2 KO male mice than in WT males (23). As a possible explanation for this finding, the present study revealed that tumor progression was associated with an increased frequency of PD-L1-positive cells in colon tissues. Furthermore, PD-L1 expression in colon tissues positively correlated with iNOS and COX-2 expressions, implying that the inflammatory milieu is involved in PD-L1 expression. These results indicate that the anti-tumorigenic effect of E2 might be associated with the immune microenvironment and cooperate with Nrf2 deficiency.

The prognostic value of PD-L1 expression in colorectal cancer (CRC) remains elusive. Recent studies using meta-analysis have shown that PD-L1 expression is a negative prognostic factor for overall survival and disease-free survival (30, 31). Although PD-L1 can be expressed on stromal, epithelial, and immune cells in CRC (32), the relative prognostic implications of these cells need to be elucidated. In the current study, we, for the first time, revealed that PD-L1 expression in colon tissues from the AOM/DSS-induced CAC mouse model was associated with colon tumor progression. Moreover, stromal and immune cells were identified as PD-L1-positive cells in colon tissue. Consistent with our findings, a recent study using the CT26 syngeneic colon tumor mouse model demonstrated that PD-L1 expression on stromal cells promoted colon cancer progression (33).

It is widely accepted that inflammation is a risk factor for carcinogenesis, as demonstrated in numerous studies (1). As a transcription factor, Nrf2 regulates the expression of anti-oxidant genes (21). Therefore, Nrf2 exerts a protective effect against inflammation-induced carcinogenesis via its ability to reduce ROS accumulation (24). In contrast, recent studies have shown that Nrf2 promotes cancer progression and metastasis (34). These controversial roles of Nrf2 in cancer are described in the literature as the “dual roles” of Nrf2 in cancer (24). In the current study, we showed that E2 more effectively suppressed inflammatory markers such as iNOS and COX-2, in the absence of Nrf2, in the cancer stage. It was associated with reduced tumorigenesis by E2. Therefore, our study highlights the critical role of E2, even in the absence of Nrf2, in inflammation-induced carcinogenesis.

A previous report has revealed that Nrf2-deficient male mice were more susceptible to AOM/DSS-induced tumorigenesis than their WT counterparts (29). However, in female mice, Nrf2 deficiency significantly attenuated tumorigenesis (based on the number of tumors >2 mm in diameter) caused by AOM/DSS treatment (28). Based on our findings in Nrf2 KO male and female mice, it could be surmised that endogenous estrogen in female mice recapitulated the effect of exogenous E2 in male mice following exposure to AOM/DSS. Compared with their WT counterparts, the reduced tumorigenesis in Nrf2 KO female and E2-treated Nrf2 KO male mice was accompanied by reduced expression of PD-L1 in colon tissues ( Figures 2 and 3 ). Moreover, Nrf2 KO female and E2-treated Nrf2 KO male mice showed decreased expression of inflammatory markers such as iNOS and COX-2, when compared with WT mice after AOM/DSS treatment. As NF-κB reportedly regulates the expression of iNOS and COX-2, E2 may play a role in suppressing the NF-κB signaling pathway in the AOM/DSS model of CAC. NF-κB is a key transcription factor of inflammation and is closely associated with colitis-associated tumorigenesis (35, 36). However, further studies are warranted to identify molecules involved in the action of E2 in the NF-κB signaling pathway.

Several inflammatory cytokines such as IFN-γ, IL-17, and TNF-α reportedly induce PD-L1 expression on tumor cells (11), and the inflammatory milieu recruits PD-L1-positive immune cells to the tumor sites (1). In addition, NF-κB has emerged as an important regulator of PD-L1 expression in the TME (12). To evaluate inflammatory conditions affecting PD-L1 expression, we examined the expression of inflammatory markers, such as iNOS and COX-2, in colon tissues. Our findings revealed that inflammatory signaling, as shown by COX-2 and iNOS expression, positively correlates with PD-L1 expression in colon tissues. Consistently, previous studies have demonstrated that COX-2 expression is associated with PD-L1 expression and immune evasion in melanoma (37) and breast cancer (38), thereby providing a rationale for evaluating COX-2 inhibition to prevent tumor immune evasion (38). In light of tumor immune evasion, our results, for the first time, suggest that the anti-tumorigenic effects of estrogen could be utilized through the suppression of PD-L1 expression. Conversely, most studies focused on hormone-sensitive cancer have shown that estrogen has a tumor-promoting effect (39). In addition, several recent studies have revealed that, in syngeneic tumor models, estrogen promotes tumor progression via the accumulation of MDSCs (16, 17), which are potent immunosuppressive cells in the TME (27). These discrepancies could be attributed to different tumor models and experimental designs. Although most studies have focused on existing tumor cells to evaluate the effects of estrogen, we investigated the effects of estrogen on colon tissues in the inflammatory phase later resulting in tumor formation by employing the AOM/DSS model of CAC and Nrf2 KO mice. Our results indicate that inadequately resolved chronic inflammation can induce immunosuppression, as represented by PD-L1 expression, and that estrogen plays a vital role in ameliorating inflammation, even in the absence of Nrf2.

In addition to being expressed on tumor cells, PD-L1 is expressed on immune cells such as MDSCs, tumor-associated macrophages, regulatory T cells, and dendritic cells (11). However, the relative contribution of these cells to immunosuppression remains poorly understood. Recent studies have revealed that PD-L1 on immune cells, rather than on tumor cells, plays a more critical role and is the relevant mechanistic target for PD-1/PD-L1 inhibitors (40, 41). Therefore, we performed IHC using colon tissues to examine which cell types expressed PD-L1. We observed that PD-L1 expression was almost exclusively found in stromal and immune cells rather than in tumor cells. Moreover, PD-L1-positive cells were significantly observed in non-tumor and tumor areas. Therefore, we quantified PD-L1-positive cells in both the tumor and non-tumor areas respectively. AOM/DSS treatment increased the frequency of PD-L1-positive cells in both tumor and non-tumor areas. After the addition of E2, the frequency of PD-L1-positive cells decreased more profoundly in Nrf2 KO mice than in WT mice. Similar to male mice, AOM/DSS treatment increased the frequency of PD-L1-positive cells in female mice. These IHC results, at least in part, showed good concordance with PD-L1 expression analyzed by Western blotting. Based on these observations, we suggest that PD-L1-positive stromal and immune cells, rather than tumor cells, might contribute to tumor progression in the AOM/DSS model of CAC.

Our study had several limitations. First, Western blot analysis did not individually demonstrate PD-L1 expression in tumor and non-tumor areas; the main reason for this was the difficulty in obtaining separate tissues from the total colon. However, the results showed good concordance with the PD-L1 expression analyzed by IHC. The second limitation is that the exact types of PD-L1-positive cells were not delineated using cell type-specific markers, although various types of PD-L1-positive cells contribute to immunosuppression depending on the context of the TME. To compensate for these limitations, we plan to further conduct T cell immunology studies in the tumor state, along with the identification of cells expressing PD-L1 in colon tissues in an AOM/DSS-induced CRC mouse model. Despite these limitations, our results undoubtedly show that the frequency of PD-L1-positive cells in colon tissues is associated with disease progression in the AOM/DSS model of CAC. To further verify the therapeutic effect of estrogen in the WT and Nrf2 KO CRC mouse model induced by AOM/DSS treatment, further studies using PD-L1 antibody should be conducted. In-depth, additional experiments are needed to further validate key findings in vitro using colon cancer cells and dissect the crosstalk between Nrf2 and PD-L1. In conclusion, our study highlights the critical role of estrogen in inflammation and PD-L1 expression, which cooperates with Nrf2 deficiency to suppress tumorigenesis.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) of the Seoul National University Bundang Hospital.

CK drafted the manuscript and performed molecular experiments. C-HS revised the manuscript and performed molecular experiments. NK designed and supervised the experiments and revised the manuscript. RN, C-HS, SC, JY, and HN performed the animal experiments. JK advised on the concept of PD-L1 in Nrf2 knockout mice. HYN assisted with the analysis of immunohistochemistry of PD-L1. RN performed the immunohistochemical analysis. JC and H-NL revised the manuscript. Y-JS provided Nrf2^+/− transgenic mice and revised the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the government of the Republic of Korea (2019R1A2C2085149).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.