Role of aldo‐keto reductase family 1 member B1 (AKR1B1) in the cancer process and its therapeutic potential

Abstract The role of aldo‐keto reductase family 1 member B1 (AKR1B1) in cancer is not totally clear but growing evidence is suggesting to have a great impact on cancer progression. AKR1B1 could participate in a complicated network of signalling pathways, proteins and miRNAs such as mir‐21 mediating mechanisms like inflammatory responses, cell cycle, epithelial to mesenchymal transition, cell survival and apoptosis. AKR1B1 has been shown to be mostly overexpressed in cancer. This overexpression has been associated with inflammatory mediators including nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB), cell cycle mediators such as cyclins and cyclin‐dependent kinases (CDKs), survival proteins and pathways like mammalian target of rapamycin (mTOR) and protein kinase B (PKB) or AKT, and other regulatory factors in response to reactive oxygen species (ROS) and prostaglandin synthesis. In addition, inhibition of AKR1B1 has been shown to mostly have anti‐cancer effects. Several studies have also suggested that AKR1B1 inhibition as an adjuvant therapy could render tumour cells more sensitive to anti‐cancer therapy or alleviate the adverse effects of therapy. AKR1B1 could also be considered as a potential cancer diagnostic biomarker since its promoter has shown high levels of methylation. Although pre‐clinical investigations on the role of AKR1B1 in cancer and the application of its inhibitors have shown promising results, the lack of clinical studies on AKR1B1 inhibitors has hampered the use of these drugs to treat cancer. Thus, there is a need to conduct more clinical studies on the application of AKR1B1 inhibitors as adjuvant therapy on different cancers.

−37 and a CCAAT box at −104 in the promoter, the AKR1B1 gene contains two Alu repeats in intron 1 and two Alu repeats in intron 4 and 9, respectively. 7 An androgen-like response element is also located at 396 to 382 nucleotides upstream of the gene. 6,8 Three osmotic response elements (OreA, OreB and OreC) are found at approximately 1 kb upstream of the transcription start site in a 132 bp region. 9 An activator protein 1 (Ap-1) binding site is positioned approximately 1100 bp upstream of the gene. 8 Thyroid receptor element (TRE) is located in the region from 1099 to 1028 upstream of the transcription start site 10 (Figure 1). AKR1B1 is translated to a monomeric in a region of 36 kD enzyme, which is located in the cytoplasm. This enzyme consumes reduced nicotinamide adenine dinucleotide phosphate (NADPH) and converts it to nicotinamide adenine dinucleotide phosphate (NADP + ) in the process of reducing aldehyde compounds to alcohol. 11 AKR1B1 plays an important role in glucose metabolism and osmoregulation and has a supportive role in the reduction of superoxides and toxic materials. 12 Because of the diverse roles in body metabolism and especially its association with NFκB, AKR1B1 has been suggested to contribute in tumorigenesis. [13][14][15] Additionally, AKR1B1 is involved in the polyol pathway. In this pathway in hyperglycaemic condition aldose reductase reduces glucose to sorbitol by consuming NADPH and later sorbitol is converted to fructose by sorbitol dehydrogenase. This was first reported by Hers in 1965. 16 NADPH is also needed for the conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH) which is an antioxidant. Concretely, some aldose reductase inhibitors have been shown to increase GSH levels. 17 The excessive sorbitol itself could play a role in osmotic stress and even the phosphorylated fructose could lead to the production of advanced glycation end products (AGEs) which eventually may increase ROS. Consequently, ectopic activation of the polyol pathway could result in different diabetic complications. [18][19][20] association with GSH does not end up here. The enzyme could also reduce lipid peroxidation products especially the ones that conjugate with GSH. 21 For example, by the action of cytokines, growth factors and lipopolysaccharides, lipid peroxidation products could ultimately be synthesized. These compounds could be converted to 4-hydroxynonenal (HNE). HNE could conjugate with GSH producing 3-glutathionyl-4-hydroxynonanal (GS-HNE), which could be converted to GS-dihydroxynonane (GSDHN). 22 AKR1B1 together with GSDHN may activate phospholipase C/ protein kinase C (PLC-PKC) pathway, which stimulates NFκB.
Hence, lipid aldehydes could affect the NFκB pathway and as a result, AKR1B1 activates the NFκB pathway by reducing GSHaldehydes. 23,24 This may prove a point that AKR1B1 could have a role in cancer promotion through NFκB activation, which has the ability to promote tumorigenicity in several cancers. 25,26 AKR1B1 is also involved in prostaglandin synthesis. In normal conditions, phospholipid is turned to arachidonic acid in a reaction, catalysed by phospholipases A2 (PLA2G) enzyme. Then, arachidonic acid is converted to prostaglandin H2 (PGH2) by the help of cyclooxygenase 1 (COX1) and cyclooxygenase 2 (COX2). AKR1B1 consumes NADPH and converts PGH2 to prostaglandin F2alpha (PGF2A).
Hence, it has been proposed that increased amounts of ROS could lead to the activation of NFκB which acts as a tissue factor (TF) for the expression of COX2. This results in the formation of excessive amounts of PGH2. On the other hand, NFκB could enhance AKR1B1 expression which causes the production of increased PGF2A from PGH2 by AKR1B1. Consequently, excessive amounts of PGF2A would lead to inflammation which could end up with increased tumorigenicity ( Figure 2). 27,28 Although the various roles of AKR1B1 have been identified in different metabolic and physiological processes, such as glucose metabolism, inflammation and prostaglandin synthesis, its true function in cancer still remains unknown. Several studies have been conducted to unveil the role of AKR1B1 in different cancers including colorectal, breast, pancreatic and hepatocellular carcinoma. In this review, we summarized the recent understandings on this topic and the improvements that could be made in cancer treatment by using AKR1B1.

| AKR1B1 in colorectal cancer
Several studies have been conducted on measuring AKR1B1 expression to find out more about its role in cancer. It has been demonstrated that AKR1B1 is expressed universally throughout the body. 29 There is still debate on how the expression of AKR1B1 affects cancer but some evidence suggests that expression of AKR1B1 in colorectal cancer (CRC) could be different depending on the stages, types and invasiveness of tumours, at least in mice models or cell lines. For example, in vivo studies have indicated higher AKR1B1 levels in invasive tumour cells in mice having colon cancer with Trp53 deletion in comparison with normal and non-invasive models. 30 In colon cancer cell lines, overexpression of AKR1B1 has been described in the metastatic SW620 cell line compared to non-metastatic SW480 cells 29 and several studies have highlighted a lower expression of AKR1B1 in SW480 and HT29. 17,31,32 In another study conducted on HT-29 and SW480, AKR1B1 mRNA expression was seen in SW480 without any protein expression; however, no AKR1B1 mRNA expression was found in HT-29 while it was seen on protein level. 33 Interestingly, in colorectal tissues, either no alteration or down-regulation of AKR1B1 has been reported, for example, by using RT-PCR, Kropotova et al for the first time reported a reduction of AKR1B1 in 10 per cent of tumour samples. 34 Besides, down-regulation of AKR1B1 in protein levels has been reported in adenocarcinoma samples. 31,35 Furthermore, a significantly different expression of AKR1B1 and S100P was found between lymph nodes categorized as Dukes' stage B groups and controls. 36 Surprisingly, Nakarai et al reported that no differential expression of AKR1B1 was observed between inflammatory, tumour and non-tumour tissues in mRNA levels. 36 Another study also showed the same results by microarray analysis. 29 Despite the fact that there is still no clear correlation between the expression of AKR1B1 and tumour creation in CRC tissues, several evidence suggest that AKR1B1 could play a role in the tumorigenesis of CRC. Accordingly, several mechanisms have also been postulated.

| Evidence for the role of AKR1B1 in inflammation
It has been proposed that ROS creation could result in the activation of inflammatory TFs such as NFκB, resulting in carcinogenesis. In this regard, it has been suggested that AKR1B1 could have a fundamental role in the regulation of ROS. 37 Consistently, ROS creation has been shown to be reduced after the knockdown of AKR1B1 in CRC. 29 AKR1B1 has also been found to be involved in the NFκB regulation. Bioinformatics analysis has demonstrated that 'regulation of cytokine production' was a significantly enriched Gene Ontology term among the AKR1B1 overexpressing samples in CRC AKR1B1 has also been found to be associated with a set of inflammatory-related genes. 29 Furthermore, silencing AKR1B1 in CRC cells has been found to cause a reduction in translocation of p65 and p50 NFκB subunits which were partially restored after renovating AKR1B1 expression.
Reduced activity and transcription of NFκB have also been reported after silencing AKR1B1. Inhibition of AKR1B1 in Caco-2 cells treated with growth factors has resulted in the reduction of NFκB. 29 Along with this, inhibition of AKR1B1 with Fidarestat resulted in the inhibition of Cox-2 and iNOS in both ApcMin/+ mice under HFD and C57BL/KsJ-db/db obese mice which contributed to low NFκB levels in cells. 38,39 NFκB binding protein has also been reported to be reduced in the metastatic liver of mice injected with HT29 or KM20 cells. 40 Another evidence that suggests AKR1B1 has a role in CRC inflammation is the notion that AKR1B1 plays a role in the synthesis of prostaglandins. In CRC, a study reported that COX2 in Caco-2 cells is required for the synthesis of prostaglandin E2 (PGE2). Fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) could induce PGE2 synthesis in Caco-2 cells via COX2. This effect has been shown to be abolished by AKR1B1 inhibition. On the other hand, after inhibition of AKR1B1, such an impact was not seen in cells without COX2. 24 Tumour necrosis factor-alpha (TNF-a) has also been elucidated to induce PGE2 and COX2 while AKR1B1 inhibition abrogated the effect in Caco-2 cells. Besides, AKR1B1 inhibition hindered the PKC and NF-κB activation induced by TNF-a. 41 Taken together, these data suggest that AKR1B1 could have a regulatory role on the inflammatory responses and the carcinogenesis through manipulation of ROS, NFκB and PGE2 synthesis in CRC.

| Evidence for the effect of AKR1B1 in cell cycle
It has been suggested that growth factor-induced ROS could activate AKT. 42 This could also result in the overexpression of G1-S regulatory proteins such as C-Myc and its downstream targets including E2F-1, cyclin-dependent kinase (CDKs) and cyclins. 40,43 The inhibition of AKR1B1 abrogates these outcomes. 43 This indicates that AKR1B1 may play a role in the progression of the cell cycle in CRC. These been proposed that AKR1B1 inhibition could arrest the proliferation of Caco-2 cells at S phase 24 and the accumulation of cells at G1 phase has been observed in HT-29, SW480 and HCT-116 cells. 43 Cyclins D1 and E, cdk4, proliferating cell nuclear antigen (PCNA), E2F and C-Myc were also suppressed following AKR1B1 inhibition. 43 Similarly, in another study, silencing AKR1B1 slowed down the progression of the cell cycle, reducing tumorigenesis in CRC as the cells transferred from G1 to S with a delay compared to normal cells. 29 It has also been reported that AKR1B1 knockdown raised the cyclin E levels in CRC with the cells in the starved state experiencing elevation in cyclin E levels compared to the cells in the released state. The study proposed that the increase in cyclin E was independent of transcriptional up-regulation as Rb phosphorylation did not change. 29 This is in contrast to the report

| Evidence for the role of AKR1B1 in mTOR pathway
Multiple lines of evidence suggest that tumour progression could be manipulated by AKR1B1 through modulating a complicated network of miRNAs, proteins and pathways. Hence, AKR1B1 inhibition might be useful in the treatment of cancer. It has been proposed that AKR1B1 inhibition by Fidarestat could prevent tumour growth induced by growth factors in CRC. Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) could reduce programmed cell death protein 4 (PDCD4) protein, a tumorigenesis suppressor, by inducing the expression of its target miRNA, mir-21, in CRC.
These growth factors could also increase ROS through the phosphorylation of PLC. Interestingly, the Inhibition of AKR1B1 has been demonstrated to down-regulate mir-21 and abrogate these effects.
Furthermore, PDCD4 could be increased in CRC by AP-1 down-regulation, a transcription factor regulating mir-21. 44 It has been reported that Forkhead box O3A (FOXO3a) expression could inhibit AP-1 activation and mir-21 expression. FOXO3A expression has also been reported to be raised after AKR1B1 inhibition in CRC cell lines. 45,46 AKR1B1 inhibition could also prevent tumorigenesis via mTOR inhibition. AKR1B1 inhibition not only could activate phosphatase and tensin homolog (PTEN) through the inhibition of phosphorylation but also could increase its expression. Thus, AKR1B1 inhibition could suppress cell proliferation by the induction of PTEN and FOXO3A which are negative regulators of PI3K/AKT/AP-1 ( Figure 3). 46 Several other studies have also indicated that AKR1B1 inhibition could reduce the phosphorylation of AKT, 38,43 and increase PKC B2 in both small and large intestines in Apc Min/+ mice under high-fat diet. 38 However, it has been shown that AKR1B1 blockage could hinder the PKC activation, triggered by either growth factors or TNF-a. 24,41 Alternatively, AKR1B1 inhibition could prevent activation of the mTOR pathway through 5' adenosine monophosphate-activated growth. AKR1B1 inhibition has also been reported to increase P53 protein, a tumour suppressor, which could inhibit mTOR activity. 47,48 It has been also suggested that ERK stimulation has been proposed to activate the mTOR pathway. 49 In this regard, it has been shown that silencing AKR1B1 in HCT-116 cells resulted in a lower proliferation, migration and wound closure as well as a lower phosphorylation of ERK1/2 in MAP kinase cascade. 29 This may be due to mTOR deactivation. These data suggest that inhibition of aldose reductase could prevent tumour growth via mTOR inhibition by different mechanisms. This could prove the usefulness of AKR1B1 inhibitors to design new drugs for target therapy in CRC.

| Evidence for the role of AKR1B1 in liver metastasis of CRC
There is evidence suggesting that AKR1B1 inhibition could hinder liver metastasis in CRC. Tammali

| AKR1B1 in breast cancer
In 2006, Saraswat et al indicated overexpression of AKR1B1 in several cancers such as breast, ovarian, cervical and rectal cancer using immunoblotting. 50 Similarly, another study reported the up-regulation of AKR1B1 in triple-negative breast cancer and the basal subtype of breast cancer cell lines. They found that AKR1B1 was expressed in basal-like breast cancer (BLBC) at the protein level while it was absent in luminal cell lines. 51 Moreover, AKR1B1 overall has shown more activity in red blood cells (RBCs) and tissues of breast cancer patients in all three grades of primary surgical and post-chemotherapy samples. 52 In contrast, in another study, AKR1B1 was reported to be suppressed in breast cancer tissue in comparison with normal breast tissue. 53 Although studies measuring AKR1B1 expression could not clearly highlight its effect on breast cancer, several evidence suggest that AKR1B1 could play a significant role in breast cancer tumorigenesis and epithelial to mesenchymal transition (EMT Taken together, these data show that AKR1B1 could be involved in a positive regulatory feedback mechanism between NFκB and Twist2 contributing to EMT. It has been suggested that EMT could cause tumour cells to obtain cancer stem cell (CSC) properties.
Therefore, AKR1B1 associated with the maintenance of CSCs and is required for tumorigenicity and metastasis of breast cancer. 51 In addition to colorectal and breast cancers, the expression level of AKR1B1 has been studied in other cancers although some have shown over-or low-expression.

Pancreas cancer
It has been demonstrated that the β2-adrenergic receptor (B2-AR) and ZEB1 suggesting an indirect interaction. 30 Interestingly, AKR1B1 overexpression was associated with decreased survival in patients with pancreatic cancer. 59 Further research is required to elucidate the exact mechanism underlying the AKR1B1 role in tumour progression in pancreatic cancer.

Lung cancer
There is evidence suggesting that AKR1B1 could promote tumour progression in lung cancer. For example, AKR1B1 up-regulation has been seen in lung cancer. 60  hepatoma cells to become more sensitive to 3-deoxyglucosone and glyceraldehyde, suggesting a role for AKR1B1 in cancer resistance in hepatoma cells. 62 The up-regulation of AKR1B1 in human HCC tissues was also demonstrated first in 2004. 63 Later, in 2018, AKR1B1 expression was reported to increase in HCC gradually from 4-month nodules to 17-month tumours in rat models of HCC. 64 The elevated AKR1B1 expression in HCC was also accompanied by other observations. Ectopic expression of AKR1B1 in the Hep2G cell line has been demonstrated to increase cell proliferation, migration, invasion, colony formation and wound healing whereas suppression of AKR1B1 caused the opposite effects. 65 In contrast to these data, a study conducted in 2015 reported that the expression of AKR1B1 in primary HCC tissues diminished in comparison with non-tumour tissues as its promoter was heavily methylated. 66 Overexpression of AKR1B1 has been indicated to trigger the AKT/mTOR signalling pathway through interaction with the AKT1 kinase domain. It increased 'Warburg effects, lactate production, oxidative stress and inflammation' resulting in tumorigenicity in HepG2 cells. In the same study, the reduction of AKR1B1 led to a decrease in AKT/mTOR signalling and cancer development in mice.
They suggested that due to the increased activity of the polyol path- sequence has been identified. These data suggested that T3 induced AKR1B1 expression is regulated by TR/TRE. 10   Thus, AKR1B1 could be involved in the initiation of endometrial cancer through modulating inflammation. 23,68 Adrenocortical carcinomas Excessive amounts of AKR1B1 has been seen in normal human adrenal tissue. 14 However, AKR1B1 expression has been demonstrated to be reduced in adrenocortical carcinoma (ACC), being less than adrenocortical adenomas and Cushing's hyperplasia. It has been proposed that cyclic adenosine monophosphate (CAMP) could regulate the expression of AKR1B1 in adrenocortical cells. Forskolin, a CAMP synthesis activator, could increase AKR1B1 expression. 14 The tissue factor cAMP-responsive element-binding protein (CREB), adrenocorticotropic hormone (ACTH) and protein kinase A (PKA) activity induced by cAMP had decreased in ACC. [69][70][71] The mechanism underlying AKR1B1 pathogenesis in ACC has not been established yet.
However, inhibition of aldose reductase has been reported to cause elevated levels of HNE which could increase phosphorylation of CREB and cell proliferation. [71][72][73] One hypothesis might be that HNE could form adducts in DNA, proteins or lipids of the body, important in cancer induction. 74 Further research is needed to unveil its accurate mechanism in ACC.

| FUTURE PER S PEC TIVE S
DNA methylation has been presented as a diagnostic biomarker for cancer detection with the advent of FDA approved tests such as Epi proColon and Cologuard, which could screen methylation of SEPT9, NDRG4 and BMP3 in CRC. [75][76][77][78] To find diagnostic, prognostic and therapeutic biomarkers with a higher performance for cancer, AKR1B1 has been chosen as a subject of study by several researchers. Although AKR1B1 expression has been found to be associated with tumour size in CRC, 35 more evidence is needed to support AKR1B1 expression as a CRC biomarker. In addition to gene expression, hypermethylation especially in the gene promoter, has been widely suggested as a diagnostic biomarker. 79,80 For example, AKR1B1 has been shown to be highly methylated in CpG islands of its promoter, involved in dysregulation mechanisms of prostaglandin-endoperoxide synthase. 81,82 Hypermethylation of AKR1B1 and its negative correlation with mRNA expression have been displayed by in silico studies. 29,83 Consistent with these data, in a study using and CRC tissues. The same study has also analysed GSE68060 dataset in which the AUC of AKR1B1 was reported to be 0.954 alongside a 98 per cent value for the beta-adducin (ADD2) gene. This suggests that methylation of these two genes could be used as a biomarker for screening and diagnosis of CRC. 83 On the contrary, AKR1B1 has been reported not to be suitable as a diagnostic biomarker for detecting lymph node metastasis as no significant differential expression between control and Dukes stage c group samples has been detected. 36 In addition to AKR1B1, AKR1B10 has also been investigated as a potential prognostic biomarker for CRC. It has been suggested that lower AKR1B1 and higher AKR1B10 expression indicate a good prognosis for this cancer and vice versa. 29 AKR1B1 could also be served as a diagnostic biomarker for breast cancer. For example, AKR1B1 promoter has been reported to be highly methylated in breast cancer tissues. [84][85][86] Besides, a study has demonstrated that AKR1B1 methylation occurred specifically in epithelial breast cell lines. 85 Another study has indicated that promoter hypermethylation of AKR1B1 and TM6SF1 could be used to detect breast cancer with an AUC of 0.986. 86 Furthermore, although it is proposed that the methylation rate in nipple fluid is less than tumour tissues, researchers have been able to differentiate cancerous nipple fluid samples from healthy ones by analysing methylation of a gene panel including AKR1B1, ALX1, RASSF1A and TM6SF1. 87 The limitation of this study was the selection of cases with different age groups in tumour and controls samples although there are no clear reports correlating of methylation and age in breast cancer. 86,87 Additionally, hypermethylation of AKR1B1 has been observed in independent her2+ breast tumours in comparison with normal breast tissues. 84 In ductal and lobular breast cancer, however, no correlation between cancerous and normal tissues in Oncomic analysis has been observed. 86 This is also some evidence suggesting AKR1B1 as a putative biomarker for hepatoma. 88 Besides, there is a negative correlation between the ratio of tumoural AKR1B1 expression to its normal tissue expression and liver cirrhosis. 88 Altogether, these data suggest that AKR1B1 methylation has the potential to be used as a diagnostic biomarker in breast cancer and CRC although further research with higher sample sizes is needed to provide more valid data.

| B I OMARK ER FOR PRED I C TI ON
Anti-cancer drug resistance is still one of the major concerns in the treatment of cancer. Drug resistance occurs in two ways; either poor initial response is seen because of the intrinsic resistance before exposing cells to drugs or through a good initial response followed by a poor outcome in which cells have acquired resistance against the drug later in the process of the treatment. The intrinsic drug resistance has been suggested to be more related to the alterations in drug breakdown, interactions of the drug with its target, transpor- the level of cytotoxicity in cells with elevated AKR1B1 levels. In addition, N-acetyl-cysteine, a substance that could induce GSH production has shown the cells to become more resistant to glyceraldehyde and diacetyl. These data propose that 2DG glyceraldehyde and diacetyl could kill tumour cells by lowering the amount of GSH, however, AKR1B1 depletion may provide more NADPH for the synthesis of GSH and this may promote cell resistance against these drugs. 17 In another study including 39 cell lines and 64 anti-cancer drugs, AKR1B1 expression alteration induced the tumour cells to become more sensitive to 23 out of 64 drugs, suggesting that AKR1B1 expression could be a putative marker for chemosensitivity prediction. 32 Table 1 summarizes the list of AKR1B1-related drugs and their effects in in vitro and in vivo studies.

| CON CLUS ION
Although pre-clinical investigations on the role of AKR1B1 in cancer and the application of its inhibitors have shown promising results, the lack of clinical studies on AKR1B1 inhibitors on cancer has hindered the use of these drugs. Thus, there is an urge to conduct more clinical studies on the application of AKR1B1 inhibitors as adjuvant therapy on different cancers.

ACK N OWLED G EM ENTS
We thank Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran and also Mashhad University of Medical Sciences, Mashhad, Iran for their support.

CO N FLI C T O F I NTE R E S T S
The authors declare no conflict of interest with respect to this research.

CO N S E NT FO R PU B LI C ATI O N
All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of the present study are available from the corresponding author upon reasonable request.