AKR1B10 accelerates the production of proinflammatory cytokines via the NF-κB signaling pathway in colon cancer

Aldo–keto reductase family one, member B10 (AKR1B10) has been reported to be involved in the tumorigenesis of various cancers. It has been reported that colorectal cancer is closely associated with chronic inflammation, but the underlying molecular mechanisms are still elusive. In our study, we evaluated the relationship between AKR1B10 expression and clinicopathological characteristics of colon cancer and showed that AKR1B10 expression was significantly correlated with the T stage and clinical stage of colon cancer. Knockdown of AKR1B10 significantly decreased the expression of the inflammatory cytokines IL1α and IL6 induced by lipopolysaccharide by inhibiting the NF-κB signaling pathway. Furthermore, AKR1B10 depends on its reductase activity to affect the NF-κB signaling pathway and subsequently affect the production of inflammatory cytokines. In addition, knockdown of AKR1B10 effectively reduced cell proliferation and clonogenic growth, indicating the biological role of AKR1B10 in colon cancer. Together, our findings provide important insights into a previously unrecognized role of AKR1B10 in colon cancer.


Introduction
Colon cancer is the third most commonly diagnosed malignancy and the fourth leading cause of cancer-related death (Arnold et al. 2017). It was estimated that in 2030, there will be more than 2.2 million new cases of colon cancer and 1.1 million deaths from colon cancer worldwide (Dai et al. 2014). At present, surgery remains the primary choice for the treatment of colon cancer. Meanwhile, adjuvant therapy is applied to diminish the rates of local recurrence and to improve the rates of survival (Nelson et al. 2001). However, the five-year survival of colon cancer patients is only approximately 50% (Adam et al. 2010). Accumulating evidence has proven that the development of colon cancer is associated with chronic inflammation and postoperative infection (Terzić et al. 2010;Eberhardt et al. 2009).
Aldo-keto reductase 1B10 (AKR1B10) is an aldose reductase-like oxidoreductase of human origin that belongs to the aldo-ketoreductase superfamily (Penning et al. 2015). AKR1B10 is typically expressed in the gastrointestinal tract, including the human colon, liver, adrenal glands and small intestine. In contrast, low expression of AKR1B10 has been identified in colon cancer, stomach cancer, and head and neck cancer (Ohashi et al. 2013;Wang et al. 2017), while high expression of AKR1B10 has been reported in various solid cancers, such as breast cancer, hepatocellular carcinoma, non-small cell lung carcinoma, cervical cancer, pancreatic carcinoma and endometrial cancer (Ma et al. 2012;Sato et al. 2016;Wang et al. 2010;Chung et al. 2012;Szymanowska-Narloch et al. 2013). Of note, our tissue microarray results showed that cases with low expression of AKR1B10 are predominant in the early stages of colon cancer, while the percentage of cases with high expression of AKR1B10 is substantially increased in the late stages of colon cancer.
The NF-κB family regulates many genes involved in immune and inflammatory responses and consists of five members: NF-κB 1 (p105/p50), NF-κB 2 (p100/p52), RelA (p65), RelB and c-Rel (Hayden and Ghosh 2008). NF-κB is constitutively activated and then induces the expression of IL-1 and IL-6 to exert various protumorigenic functions in many kinds of tumor cells (Lawrence 2009). IL-1α was the first identified member of the IL-1 cytokine family, and it has pleiotropic effects in inflammation and cancer (Malik and Kanneganti 2018). Recently, researchers have also demonstrated IL-1α is an apical regulator of colon inflammation and cancer, cardiovascular disease and neural inflammation (Bersudsky et al. 2014;Freigang et al. 2013;Brough and Denes 2015). IL-6, which belongs to the IL (interleukin)-6type cytokine family, has both pro-and anti-inflammatory properties and plays a crucial role in the acute-phase and immune responses of organisms. IL-1α and IL-6 are found in most cell lines, including tumor cell lines (Heinrich et al. 2003).
In our study, we found that AKR1B10 could promote the production of the inflammatory cytokines IL-1α and IL-6 through the NF-κB signaling pathway. The aldose reductase activity of AKR1B10 was responsible for its proinflammatory effects. In addition, we also found that AKR1B10 promoted cell proliferation and colony formation of colon cancer cells.
In summary, our study explored the clinicopathological expression pattern of AKR1B10 in colon cancer and preliminarily revealed the relationship between AKR1B10 and inflammation, proliferation and the colony formation of colon cancer cells.

Cell culture
Colon cancer HT-29 cells were obtained from the Cancer Research Center of Xiamen University. Cells were cultured in DMEM supplemented with 10% FBS and maintained at 37 °C under an atmosphere of 95% air and 5% CO 2 .

Oncomine database analysis
An online microarray database-oncomine (http:// www. oncom ine. org) was used to compare AKR1B10 mRNA levels between colon cancer tissues and normal tissues. The thresholds were set as follows: p-value: 0.0001; fold change: 2; gene rank: 10%; analysis type: cancer vs. normal analysis.

Immunohistochemical analysis
The expression of AKR1B10 in paraffin-embedded tumor samples was determined by immunohistochemistry (IHC) staining of tissue microarrays. IHC was performed using a standard avidin-biotin-peroxidase method. The TMAs were deparaffinized in xylene and then dehydrated in gradient concentrations of ethanol. The samples were placed in a microwave oven for 5 min with preheated sodium citrate buffer. After rinsing with distilled water in 3% hydrogen peroxide for 10 min, the sections were incubated with normal goat serum for 10 min at room temperature to eliminate nonspecific staining. The sections were incubated with rabbit anti-AKR1B10 antibody overnight at 4 °C and then incubated with the secondary antibody at 37 °C for 30 min. Subsequently, the sections were incubated with the avidin-biotin-peroxidase complex for another 10 min. Then, the sections were stained with 3,3-diaminobenzidine (DAB) for 1-5 min and counterstained with hematoxylin. Negative controls were obtained by replacing the primary antibody with PBS. Images were acquired on Motic VM1 and processed with Motic DSAssistant Lite software. The immunohistochemical staining was blindly scored by two pathologists.

AKR1B10 knockdown by stable transfection
The shRNA primers were designed according to the pLV-RNAi system, and the sequences used were as follows: shAKR1B10#1: GAA CAA ACC TGG ACT GAA ATA;shAKR1B10#2: GGT TCT GAT CCG TTT CCA TAT. The lentivirus vector pLV-AKR1B10-puromycin or plvcontrol-puromycin was transfected into 293 T cells together with the auxiliary plasmids pMDLg/pRRE, pVSV-G and pRSC-Rev to package the lentivirus. After infection with the lentivirus, shAKR1B10 cells and shcontrol cells were screened out by adding puromycin to the media.

Overexpression and mutant plasmids establishment
Human AKR1B10 cDNA was used to design the PCR primers as follows: AKR1B10-N-F:AGA GAA TTC GGA TCC GCC ACG TTT GTG GAG CTC AGT ACCAA; AKR1B10-N-R:TGG CTC GAG CCC GGG TCA ATA TTC TGC ATT GAA GGG ATAGT. The eukaryotic expression plasmid pLV-N-Flag-AKR1B10 of AKR1B10 was obtained by LIC ligation. Using this plasmid as a template, pLV-N-Flag-AKR1B10 (K125L) was obtained by using AKR1B10 enzyme active point primers: AKR1B10-K125L-F:CCT TTT CCC CTT AGA TGA TAA AGG;AKR1B10-K125L-R:CCT TTA TCA TCT AAG GGG AAA AGG . The plasmids established above were transfected into the AKR1B10 knockdown cell line.

Western blot analysis
Colon cancer HT-29 cells were lysed in RIPA lysis buffer supplemented with the protease inhibitor PMSF to extract total protein. Nuclear lysates were isolated using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Protein lysates were separated on 10% or 13% SDS-PAGE gels. Then, the proteins were transferred to PVDF membranes and blocked with 5% BSA for 1 h. Afterward, incubation was carried out with the primary antibodies overnight at 4 °C. After that, the cells were washed with TBST for 10 min, and this process was repeated at least three times. Next, incubation was carried out with the secondary antibodies for 1 h at room temperature. The membranes were washed for 10 min each three times. Immunoreactive bands were detected using electrochemiluminescence.

Cell proliferation and cell colony formation assays
The viability of the cells was measured by using a cell counting kit (CCK8) assay. For the CCK-8 assays, 5000 cells in 100 μl of medium were seeded into each well of a 96-well plate. Cells were cultured for 24, 48 and 72 h at 37 °C. Finally, 10 µl CCK-8 sodium was added to each well and incubated for 2 h at 37 °C. Then, the optical density was measured at a wavelength of 450 nm.
For the cell colony formation assay, 50, 100, and 200 cells in 5000 μl of medium were seeded into 6 cm plates. The culture medium was changed every three days. When visible clones appeared after 2 -3 weeks, the medium was removed, the plates were washed twice with PBS, and air-dried for 10 min. Then, 100% formaldehyde was used to fix the cells at room temperature for 15-20 min. Finally, the plates were stained with Giemsa for 10 min. Images were taken, and the number of colonies formed was counted.

Statistical analyses
Immunohistochemical AKR1B10 staining was compared among the subgroups by Kaplan-Meier analysis. P-values of < 0.05 were considered statistically significant. For the statistical analyses, we used R for Windows.

AKR1B10 is downregulated in colon cancer
The Oncomine database was used to compare AKR1B10 mRNA expression levels between colon cancer tissues and normal tissues. The Hong colorectal cancer dataset indicated that the AKR1B10 mRNA levels were significantly reduced in colon cancer (Fig. 1a). This is in accordance with previous reports that AKR1B10 is underexpressed in colon cancer and gastric cancer. Tissue microarray analysis of 80 colon cancer samples confirmed that AKR1B10 expression was considerably reduced in colon cancer tissues compared with normal tissues (P < 0.01, Fig. 1b-c). In addition, AKR1B10 was localized mainly in the cytoplasm (Fig. 1c).
After analyzing the correlation of AKR1B10 expression in colon cancer tissues with clinicopathological 1 3 Fig. 1 Reduced expression of AKR1B10 in colon cancer. a AKR1B10 expression in the Hong colorectal cancer dataset from Oncomine. b AKR1B10 expression was analyzed by immunohisto-chemical staining based on a TMA containing 80 colon cancer specimens and 80 noncancerous tissue specimens. c Representative images of immunohistochemical staining of AKR1B10 characteristics (Table 1), we found that AKR1B10 expression was significantly associated with the T stage and clinical stage of colon cancer but not with other clinicopathologic variables, such as age, sex, position and differentiation degree of the cancer (representative pictures of the IHC along with H&E and T stage are shown in Figure S1-S3). In our study, although low expression of AKR1B10 is predominant in colon cancer, the proportion of tumors with high expression of AKR1B10 increased with the progression of T stage and the clinical stage. These results might indicate a role of AKR1B10 in the oncogenesis and progression of colon cancer.
Accumulating studies have confirmed that LPS can induce the expression of not only inflammatory cytokines in macrophages but also in tumor cells (Tannahill et al. 2013;Yang et al. 2015). Due to intestinal mucosal permeability and postoperative bacterial displacement, the patient's LPS content is increased. LPS-induced inflammatory cytokines are critical for tumor development. Therefore, we also determined the effect of AKR1B10 on LPS-induced inflammatory cytokine expression (Fig. 2c). Compared to the control cells, AKR1B10 knockdown obviously inhibited the mRNA levels of the cytokines IL-1α and IL-6 when the cells were stimulated with LPS. Moreover, AKR1B10 rescue reversed the inhibition of the expression of inflammatory factors in shAKR1B10 cells stimulated with LPS. Collectively, these results suggested that AKR1B10 promotes the expression of IL-1α and IL-6 in HT-29 cells (the effects of AKR1B10 on inflammatory cytokine expression in HCT8 cells are shown in Fig. S4).

The aldose reductase activity of AKR1B10 is required for the promotion of inflammatory cytokines production
Accumulating evidence has revealed that the aldose reductase activity of AKR1B10 is linked to its physiological function (Gallego et al. 2007). In order to explore whether the aldose reductase activities of AKR1B10 is essential for the production of inflammatory cytokines, Oleanolic Acid (OA), an specific and potent inhibitor of AKR1B10, was used to inhibit the aldose reductase activity of AKR1B10. As shown, the mRNA levels of IL1α were declined in LPSinduced HT-29 cells after treatment of OA (Fig. 3a).
To further ensure this hypothesis, the AKR1B10 K125L mutant stably overexpressed in shAKR1B10 cells were used (Fig. 3b). Lys-125 site of AKR1B10 is a key residue to attain a high catalytic efficiency with all-trans-retinaldehyde (Gallego et al. 2007). The results showed that replenishment with the AKR1B10 K125L mutant could not effectively rescue the mRNA expression of IL-1α and IL-6 compared to replenishment with wild-type AKR1B10 (Fig. 3c). These results indicated that the aldose reductase activities of AKR1B10 play an important role in regulating the production of IL-1α and IL-6.

AKR1B10 promotes the production of inflammatory cytokines via the NF-κB pathway
A large number of studies have shown that NF-κB and STAT3 play important roles in the regulation of the expression of certain inflammatory cytokines (Hart et al. 2011;Dong et al. 2014). We explored whether AKR1B10 affected NF-κB or STAT3 to regulate the expression of inflammatory cytokines. Our results showed that it is hard to determine whether AKR1B10 affects the STAT3 pathway, which needs to be further explored (Fig. 4a). However, we found that knockdown of AKR1B10 led to a decrease in the phosphorylation level of the total P65 protein, and rescue of AKR1B10 reversed this change (Fig. 4a). Nuclear localization of P65 was dramatically decreased in shAKR1B10 cells and increased in Flag-AKR1B10 (Rescue) cells, especially when the cells were treated with LPS ( Fig. 4b-c). Replenishment of wild-type AKR1B10 reversed the effect of shAKR1B10 and promoted the shift of p65 from the cytoplasm to the nucleus, while replenishment with the AKR1B10 K125L mutant did not (Fig. 4d), which indicated that the aldose reductase activity of AKR1B10 is essential for activating the NF-κB signaling pathway.
To test the effect of NF-κB on the production of IL-1α and IL-6 in HT-29 cells, we treated the cells with BAY 11-7082, an inhibitor of NF-κB, to block the phosphorylation of IκBα and found that the production of IL-1α and IL-6 was inhibited when NF-κB was inactivated (Fig. 4e). Taken together, these data demonstrated that AKR1B10 induced the production of the inflammatory cytokines IL-1α and IL-6 by activating the NF-κB signaling pathway.

AKR1B10 promotes cell proliferation and clonogenic growth
To clarify the relationship of AKR1B10 and cell growth in human colon cancer, CCK8 and clonogenic assays were performed in HT-29 cells. As shown, AKR1B10 knockdown inhibited cell proliferation and AKR1B10 rescue promoted cell proliferation in HT-29 cells (Fig. 5a). Cell proliferation and tumorigenesis were further determined and the results revealed that the efficiency of colony formation was significant reduced in AKR1B10 knockdown Fig. 2 Effects of AKR1B10 on inflammation cytokine expression in HT-29 cells. The mRNA levels were quantitated by RT-PCR normalized to GAPDH. (*P < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). a The AKR1B10 knock down cell lines (shAKR1B10#1, shAKR1B10#2) and AKR1B10 rescue cell lines (FlagAKR1B10-Rescue). b The mRNA levels of IL1α and IL6 in shctrl, shAKR1B10 (labeled as #1 and #2, respectively) and Fla-gAKR1B10 (Rescue) cells without any stimulation. c The mRNA levels of IL1α and IL6 in shctrl, shAKR1B10 (labeled as #1 and #2, respectively) and FlagAKR1B10 (Rescue) cells stimulated with LPS (100 μg/ml, 1 h) cells and clearly increased in AKR1B10 rescue cells (Fig. 5b).

Discussion
AKR1B10 is an NADPH-dependent monomeric reductase that can reduce a variety of aldehydes. Moreover, retinoidbinding sites, especially at position 125, are determining factors for the all-trans-retinaldehyde specificity of AKR1B10 (Gallego et al. 2007). AKR1B10 is highly expressed in normal colon tissue, whereas it has limited expression in colon cancer tissues. Of note, our results demonstrated that despite the generally low expression of AKR1B10 in colon cancer, the expression of AKR1B10 increased with the progression of colon cancer. In the intestine and adrenal gland, the biological function and the role of AKR1B10 in tumor development and progression are reported as follows. AKR1B10 may act as an important cell survival protein by modulating lipid synthesis, mitochondrial function and oxidative status, as well as carbonyl levels (Wang et al. 2009). As an important protective enzyme of colonic epithelial cells, AKR1B10 protects host cells from DNA damage induced by electrophilicarbonyl compounds in colon cells (Zu et al. 2017).
The AKR1B10 gene contains several putative regulatory motifs of NF-κB (Nishinaka et al. 2011). NF-κB dimers are retained in the cytoplasm by specific inhibitors, IkBs, in most types of cells. NF-κB can be activated by exposure of cells to LPS or other inflammatory cytokines (Baldwin 1996). Phosphorylation of P65 and translocation of NF-κB dimers from the cytoplasm to the nucleus are favorable evidence for the activation of NF-κB. Meanwhile, the activation of NF-κB has long been linked to the production of inflammatory cytokines (Liu et al. 2015). The expression of inflammatory factors plays an important role in the development of colon cancer (Gout and Huot 2008). IL-1α has an antitumor effect by boosting antitumor immunity at low levels. However, increased release of IL-1α maintains a close link with protumorigenic effects, since it can promote the survival and proliferation of cancer cells (Ozawa et al. 2019). Previous studies have also shown that elevated IL-6 activates early immunity to exert an antitumor effect (Rakhesh et al. 2012). Dysregulation of the IL-6 cytokine is considered an important step in inducing and maintaining several diseases, such as rheumatoid arthritis, inflammatory bowel disease, osteoporosis and various types of cancer. In addition, LPS is the main component of the outer membrane of Gram-negative bacteria. It can induce severe sepsis systemic inflammation Fig. 3 Aldose reductase activity of AKR1B10 is essential for the production of inflammatory cytokines IL1α and IL6. The mRNA levels of IL1α and IL6 were quantitated by RT-PCR normalized to GAPDH. a HT-29 cells were incubated with 100 μg/ml LPS and/or 10 μM OA for 1 h. b Replenish the AKR1B10 K125L mutant into shAKR1B10 cells. This cell line was named FlagAKR1B10 (K125L) (Rescue). c FlagAKR1B10 (K125L) (Rescue) cells were treated with or without100μg/ml LPS for 1 h Fig. 4 AKR1B10 induced the production of inflammatory cytokines via the NF-κB signaling pathway. a Western blot analysis of STAT3 expression and p65 expression in whole cell lysates from shctrl, shAKR1B10#1 and FlagAKR1B10 (Rescue) cells. b Western blot analysis of p65 expression in nuclear and cytosolic lysates in shctrl, shAKR1B10#1 and FlagAKR1B10 (Rescue) cells. c Western blot analysis of p65 expression in nuclear and cytosolic lysates in shc-trl and shAKR1B10#1 cells after treatment with 100 μg/ml LPS for 1 h. d Western blot analysis of p65 expression in nuclear and cytosolic lysates in shctrl, FlagAKR1B10 (Rescue) and FlagAKR1B10 (K125L) (Rescue) cells after treatment with 100 μg/ml LPS for 1 h. e The mRNA levels of IL1α and IL6 in HT-29 cells. HT-29 cells were stimulated with LPS (100 μg/ml, 1 h) in the presence or absence of 100 μM BAY 11-7082, an inhibitor of NF-κB signaling as well as postoperative infectious complications after colon surgery.
In conclusion, we have shown for the first time that the expression of AKR1B10 is significantly correlated with T stage and clinical stage in human colon cancer. The colon cancer HT-29 cells were used as a model system to reveal the relationship between AKR1B10 and the production of inflammatory cytokines. In the present study, we showed that AKR1B10 activates NF-κB and promotes the production of IL-1α and IL-6 in colon cancer cells. Such effects are tightly associated with the aldose reductase activities of AKR1B10, which was shown by the decreased expression of IL-1α and IL-6 and the downregulation of nuclear localization of P65 in AKR1B10 K125L mutant rescue cells. The effects of AKR1B10 on cell proliferation and clonogenic growth further support a role of AKR1B10 in colon cancer. More research needs to be performed to address the role of AKR1B10 in the inflammatory microenvironment of colon cancer in our future work.