Aldose reductase interacts with AKT1 to augment hepatic AKT/mTOR signaling and promote hepatocarcinogenesis

Marked up-regulation of aldose reductase (AR) is reportedly associated with the development of hepatocellular carcinoma (HCC). We investigated how aberrantly overexpressed AR might promote oncogenic transformation in liver cells and tissues. We found that overexpressed AR interacted with the kinase domain of AKT1 to increase AKT/mTOR signaling. In both cultured liver cancer cells and liver tissues in DEN-induced transgenic HCC model mice, we observed that AR overexpression-induced AKT/mTOR signaling tended to enhance lactate formation and hepatic inflammation to enhance hepatocarcinogenesis. Conversely, AR knockdown suppressed lactate formation and inflammation. Using cultured liver cancer cells, we also demonstrated that AKT1 was essential for AR-induced dysregulation of AKT/mTOR signaling, metabolic reprogramming, antioxidant defense, and inflammatory responses. These findings suggest that aberrantly overexpressed/over-activated hepatic AR promotes HCC development at least in part by interacting with oncogenic AKT1 to augment AKT/mTOR signaling. Inhibition of AR and/or AKT1 might serve as an effective strategy for the prevention and therapy of liver cancer.


INTRODUCTION
Aldose reductase (EC1.1.1.21, AKR1B1, AR) is a member of the aldo-keto reductase (AKR) protein family, which plays important roles in nuclear receptor signaling, inflammatory responses, osmoregulation, endobiotic and xenobiotic detoxification, hormone synthesis and action, cellular metabolism and reproduction etc. [1]. For glucose metabolism, AR serves as the first and the rate-limiting enzyme for the polyol pathway (PP) to reduce glucose to sorbitol, while sorbitol is further oxidized by sorbitol dehydrogenase (SDH) to generate fructose [2].
In the liver, AR was found to be transiently expressed during embryogenesis [3]. In adult animals, hepatic AR expression or activity is barely detectable or absent [3,4]. A number of recent studies, nonetheless, have shown that hepatic AR can be significantly induced and activated under a variety of stress conditions or in diseased livers.

Research Paper
Oncotarget 66988 www.impactjournals.com/oncotarget In humans or rodents, AR and aldo-keto reductase family 1B10 (AKR1B10, also known as AR-like-1), were often among the most significantly up-regulated genes in many types of cancers including cervical cancer, colon cancer, leukemia, pancreatic cancer and hepatocellular carcinoma (HCC) [4][5][6][7][8][9][10][11]. Consistent with the transcriptomic analyses, many proteomic studies also indicated very significant elevations in the protein expression of AR and AKR1B10 in human liver cancer tissues [5,7,8,12]. More interestingly, some reports showed that AR/ AKR1B10 mRNA expression levels is an independent predictor of prognosis in HCC patients [10,11]. In spite of these studies, however, very little studies were focus on the mechanism of the aberrantly overexpressed AR/ AKR1B10 contribute to the development or progression of various types of cancers.
Potential mechanisms are aberrant overexpression/ activation of hepatic AR and/or Polyol Pathway (PP)associated overt oxidative stress and inflammation, which are believed to contribute significantly to the development of cancers [4,13,14]. Studies also suggest that inhibition of oxidative stress or inflammation is helpful with cancer prevention or treatment. For instance, trans-aldolase deficiency-induced hepatocarcinogenesis was associated with activation of AR that can be prevented by treatment with N-acetylcysteine [15]. In rats, diethylnitrosamine (DEN)-induced hepatocarcinogenesis was also associated with activation of AR and treatment with a ROS scavenger dially sulfide significantly ameliorated DENinduced HCC [16].
Aberrant overexpression/activation of hepatic AR/ PP may also contribute to lactate over-production, as in the well-known "Warburg effect" or aerobic glycolysis, whereby cancer cells exhibit increased conversion of glucose to lactate, even in the presence of sufficient oxygen [17]. Aberrant AR/PP-mediated hepatic overproduction of fructose were shown to reprogram cellular glucose-lipid metabolism to significantly affect the development of obesity, metabolic syndrome, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis [18][19][20][21], all of which are important risk factors for the development of HCC. Fructose by itself was suggested to be able to promote tumorigenesis, in part by inducing metabolic reprogramming and lactate over-production [22][23][24][25]. However, the relationship between AR and lactate-production/Warburg effect has been unclear.
In the present study, we investigated the potential roles of AR in the development of HCC. The effects of AR overexpression and AR knockdown/knockout on lactate formation, the expression of inflammatory cytokines, and the most important Warburg effect regulating pathway, the AKT/mTOR signaling pathway, were evaluated in vitro in cultured liver cancer cells and in vivo in the livers of DENinduced transgenic HCC model mice.

Overexpression of AR enhanced whereas knockdown of AR suppressed cancer cell proliferation, colony formation, and migration, invasion and wound-healing
In humans, microarray analyses identified AR and AKR1B10 mRNA up-regulated in the development of hepatitis C virus (HCV)-associated HCC [26,27]. AR mRNA ranked at the top 3% and 7% of the significantly altered genes in HCV-positive HCC and HCV-positive cirrhosis respectively, in comparison with the HCVnegative normal subjects ( Figure 1A, p < 0.001). Meanwhile, AKR1B10 mRNA ranked at the top 2% and 9% of the significantly altered genes in HCC and cirrhosis respectively (p < 0.001).
To evaluate the effects of AR overexpression or knockdown on hepatocarcinogenesis, we performed transfection studies using HepG2 or SMMC-7721 liver cancer cells with a plasmid overexpressing AR or three plasmids overexpressing shRNAs against AR (Supplementary Table 1). In HepG2 cells, AR overexpression significantly enhanced HepG2 cell proliferation ( Figure 1B), colony formation ( Figure 1C), migration and invasion ( Figure 1D), and wound-healing ( Figure 1E). In contrast, shRNA-mediated knockdown of AR significantly suppressed HepG2 cell proliferation ( Figure 1B), colony formation ( Figure 1C), migration and invasion ( Figure 1D), and wound-healing ( Figure 1E). Remarkably, inhibition the phosphorylation of AKT1 by LY294002 significantly suppressed AR-induced woundhealing ( Figure 1E) [28]. Furthermore, although small but not significant difference in wound-healing was found between the AR knockdown cells (pLV-shAR-1 transfected) and the control cells (pLV-ctrl-transfected), knockdown of AR suppressed wound-healing in EGFstimulated cells (pLV-ctrl+EGF versus pLV-shAR-1+EGF) [29]. Similar effects of AR overexpression or knockdown on cell proliferation, colony formation, migration and invasion, and wound-healing observed in SMMC-7721 liver cancer cells (Supplementary Figure 1). Together these results suggested that aberrantly overexpressed AR promote oncogenic transformation or metastasis.
In the case of Akt1/2/3 isoforms have the similar structure (Supplementary Figure 3), following experiments performed using human Akt1 as a representation of Akt family. Also previous studies of mice showed that Akt1 were more important to cell growth, whereas Akt2 mediated glucose metabolism [36][37][38], this study focused on Akt1 only. Further co-immunoprecipitation analyses indicated that AR overexpression markedly suppressed the binding of MYC-ubiquitin to AKT1, whereas this effect was not significantly in AR knockdown cells ( Figure 2D and 2E). AR overexpression thus might stabilized AKT1 in part through preventing proteosomemediated degradation of ubiquitinated AKT1.

AR interacted with the kinase domain of AKT1
To further explore the molecular mechanisms of AR stabilized AKT1 and AKT2, immunoprecipitation (IP) assays were performed. In HEK293T cells, Flag-tagged AR co-precipitated with HA-tagged AKT1, using either anti-Flag or anti-HA antibody ( Figure 3A). Furthermore, the E. coli expressing His-tagged AKT1 combined E. coli expressing GST-tagged AR but not the empty vector pGEX-4T1-GST ( Figure 3B).
In comparison with the control cells, AR overexpression increased lactate formation ( Figure  4C) and LDH activity ( Figure 4D), AKT1 knockdown by siRNA (siAKT1) or inactivation by LY294002 treatment significantly diminished AR overexpressioninduced lactate formation and LDH activity increasing. Meanwhile, AR knockdown suppressed lactate formation and LDH activity, but AKT1 overexpression significantly restored AR knockdown-induced lactate formation and LDH activity decreasing.

Liver-specific AR overexpression tended to promote whereas Ar deficiency tended to suppress DEN-induced HCC
To examine the effects of AR regulated hepatic Akt/ mTor signaling, lactate formation, inflammatory response gene expression and liver cancer development in vivo, HCC was induced in 2-wk old WT/FVB, TG2/FVB, TG1/ FVB, WT/B6 and KO/B6 [47,48] male mice by a single injection of DEN at the dosage of 25 mg/kg body weight. In FVB mice, DEN-treated AR-overexpressing mice (TG2/ FVB, TG1/FVB) had significantly higher tumor incidence (% mice with tumors > 1 µm), visible tumor number per mouse, maximal tumor size and accumulated tumor size (in diameter, mm) than the DEN-treated control mice (WT/FVB) ( Figure 5), but not in body or liver weight (Supplementary Figure 5). Conversely, a significant amelioration in tumor incidence, visible tumor number per mouse, maximal tumor size and accumulated tumor size was observed in Ar deficient B6 mice (KO/B6) as compared to the control mice (WT/B6) ( Figure 5), with a few minor exceptions. Together, these data suggested that liver-specific AR overexpression promote whereas Ar deficiency/knockdown suppress HCC tumorigenesis or progression.

Significant alterations in Akt/mTor signaling, lactate formation, and Tnfα/Il-6 mRNA expression in the liver tissues of ARoverexpressing transgenic and Ar knockout mice
As demonstrated by immunohistochemical (IHC) analyses, liver-specific AR overexpression significantly increased hepatic Akt1 expression, whereas Ar deficiency significantly reduced hepatic Akt1 expression in DENtreated mice ( Figure 6A). Moreover, AR overexpression increased cell proliferation marker Ki67, whereas Ki67 significantly suppressed in Ar deficient mice ( Figure 6A). In AR overexpression mice exposed to DEN for 34 wk, hepatic protein levels of mouse p S473 -Akt1, total Akt1/ Akt2, p S256 -FoxO1, mTor, Hif1α, Pkm2 and Srebp significantly increased ( Figure 6B). Conversely, in Ar deficient mice exposed to DEN for 42 wk, the assayed proteins significantly reduced, except FoxO1.
Also consistent with the in vitro studies, AR overexpression increased serum and hepatic lactate concentration, liver LDH and ALT activities, and hepatic Tnfα/Il-6 mRNA expression ( Figure 6C-6F and Supplementary Figure 6), although the differences in hepatic lactate, LDH activity and Tnfa were not significantly of 30 wk. Conversely, Ar knockout decreased serum and hepatic lactate concentration, liver LDH and ALT activities, and hepatic Tnfα/Il-6 mRNA expression ( Figure 6C-6F and Supplementary Figure 6), with the exception that the differences in Tnfα mRNA, serum and hepatic lactate were not significantly of 38 wk. In general, these data indicated that liver AR expression regulate Akt/mTor signaling, lactate formation, and Tnfα/Il-6 expression in vivo.
Oncotarget 66996 www.impactjournals.com/oncotarget AKT1 ( Figure 2D and 2E) and eventually significant augmentation of AKT/mTOR signaling (Figure 2A). As a consequence of its interaction with AKT1, the overexpressed AR augmented AKT/mTOR signaling (Figures 2A, 2C and 6D) and tended to enhanced lactate formation and hepatic inflammation (Figures 4 and 6). Conversely, AR knockdown suppressed lactate formation and inflammation. In cultured HepG2 cells, we further demonstrated that AKT1 was essential for AR-induced dysregulation of AKT/mTOR signaling, metabolic reprogramming, antioxidant defense and inflammatory responses (Figure 4). We also clearly demonstrated that liver-specific AR overexpression leads to abnormal augmentation in hepatic AKT/mTOR signaling ( Figure 6A) and enhanced HCC development ( Figure 5). By contrast, oncogenic AKT/mTOR signaling and HCC development appears to be significantly ameliorated in mice deficient in Ar, with a few minor exceptions. Together, these data suggest that aberrantly overexpressed/over-activated hepatic AR promotes HCC development, at least in part by interacting with the oncogenic AKT1 to augment AKT/ mTOR signaling.
Cancer cell metabolism is characterized by the so-called Warburg effects, the manifestations of which include enhanced glucose uptake and glycolysis, reduced oxidative phosphorylation and increased lactate secretion [53]. What might have long been overlooked, however, is the fact that the high intracellular glucose present in cancer cells very likely will trigger the overexpression of AR and/or over-activation of PP. It has been estimated that when glucose is abundant, more than 30% of glucose can be channeled into the AR/PP, which can lead to the over-production of fructose [2]. In mammalian cells, fructose also has a tendency to be converted into lipid, uric acid and lactate [54]. In this regard, it is very likely that overexpression of AR/over-activation of PP in cancer cells contribute significantly to cancer-associated metabolic reprogramming, in part by increasing synthesis of lipid, lactate and uric acid. In this investigation, we clearly demonstrated that AR overexpression promotes, and AR inhibition inhibits, lactate formation. The increase in lactate secretion from cancer cells, on the other hand, might be attributable to two distinct mechanisms: 1) high glucose directly activates AR/PP leading to the overproduction of fructose and lactate; 2) overexpressed AR interacts with AKT1 to augment AKT/mTOR, HIF1a and PKM2 signaling, eventually leading to increased flux through the aerobic glycolysis to enhance lactate formation. Lactate secretion due to the over-activation of AR/PP or the augmented AKT/mTOR signaling due to AR-AKT1 interaction in cancer cells therefore might account for a significant portion of the total lactate formation, which was attributed mostly to the Warburg effects previously.
More interestingly, some reports showed that AR/ AKR1B10 overexpression promotes the development of resistance against various chemotherapeutic drugs [4,55,56]. In addition, various studies were revealed that increased expression of AR and AKR1B10 is involved in carcinogenesis and drug resistance [10,11,55], and AR/ AKR1B10 inhibitors could be potentially effective drugs for cancer therapeutics [55,56]. Zopolrestat, an AR/AKR1B10 inhibitor, was found to provide additional therapeutic effects in liver cancer [57]. Epalrestat, an AKR1B1 inhibitor, significantly suppresses cancer stem cell properties, tumorigenicity, and metastasis of basal-like breast cancer cells through regulating the NF-κB pathway [58]. Consistent with previous observations, AR knockdown was found to increase susceptibility to chemotherapeutic agents, while AR expression led to tumor cell resistance to anticancer drugs (Supplementary Figure 7). Therefore, suppression of AR by inhibitors or siRNAs has the potential to serve as an adjuvant therapeutic strategy for cancers [59]. Until very recently, however, no AR inhibitor was evaluated in clinical human cancer therapy. Fidarestat, another AR inhibitor, has already been passed through the FDA's Phase-III clinical trials and has proven safe for human use, without irreversible toxicity [4]. Thus, this drug could soon be used for various cancer therapies, though clinical studies of combination therapies using known chemotherapeutic drugs with AR inhibitors/siRNAs are needed to further assess clinical toxicity and risks.
In summary, we demonstrated in this investigation that when overexpressed in liver cells, AR may mediate over-activation of PP, causing over-production of fructose, lactate, and ROS and altered expression of inflammatory response genes. Overexpressed hepatic AR may also interact with AKT1 to augment AKT-mTOR signaling, further promoting metabolic reprogramming and dysregulation of antioxidant defense and inflammatory responses. Overactivation of the polyol pathway and AR-augmented abnormal AKT-mTOR signaling may act synergistically to promote tumorigenesis in the liver. More importantly, interfering with AR/PP expression/activation may be an effective adjuvant strategy for clinical cancer therapy.

Clinical mRNA expression analysis
Clinical Akr1b1 or Akr1b10 mRNA expression were analysed in cirrhosis and HCC on www.oncomine. org. Searching "Akr1b1" or "Akr1b10" gene expression in Mas Liver Database (GSE14323) with the following parameters "clinical specimen" in sample type, "mRNA" in Data type, "Liver cancer" in cancer type.

Liver-specific human AR overexpressing transgenics and Ar deficient knockout mice
The liver-specific human AR-overexpressing transgenic FVB (TG1/FVB and TG2/FVB) mice were generated by Dan Song at Yun-qing Yang's lab of Xiamen Oncotarget 66997 www.impactjournals.com/oncotarget University. The liver-specific human AR-overexpressing transgenic FVB (TG1/FVB and TG2/FVB) mice, Ar knockout C57BL/6 mice (KO/B6) and their controls (WT/ FVB or WT/B6) were also kind gifts from Prof. Yun-qing Yang and only used for this project.
Mice were bred and maintained under a standard 12-12 h light-dark cycle, and were fed standard rodent chow and water ad libitum, and housed in the barrier facility of the Laboratory Animal Center, Xiamen University. All animal experiments were performed according to the protocols approved by the Institutional Animal Use and Care Committee of Xiamen University, China.

Hepatocarcinoma (HCC) induction in mice
For the induction of HCC, the male mice of liverspecific human AR-overexpressing transgenic FVB (TG1/ FVB and TG2/FVB), Ar knockout C57BL/6 (KO/B6) and their controls (WT/FVB or WT/B6) were injected intraperitoneally with diethylnitrosamine (DEN) at 25 mg/kg body weight at the age of 2 wk (Cat# 049k1613v, Sigma-Aldrich, St. Louis, MO, USA) [60]. DEN-treated TG/FVB and WT/FVB male mice were sacrificed either 30 or 34 wk of age, while DEN-treated KO/B6 and WT/ B6 male mice were sacrificed either 38 or 42 wk of age. Liver lobes were photographed and tumors > 1 mm in diameter on liver surface were counted. The diameters were measured using a vernier caliper. Liver tissues were also dissected for further analyses. The phenotype data of two independent DEN-induced HCC mice models were analysed together in Figure 5. All experimental procedures involving animals were performed in accordance with the animal protocols approved by the Laboratory Animal Center of Xiamen University.

Other procedures
All of the other procedures are established standard techniques and are described in the Supplementary Files.

Statistical analyses
All statistical analyses were performed with the GraphPad Prism 5.0 software. Values are expressed as the means ± SEM. The Student's t-test (two-tailed) for pair-wise comparisons. A probability value (p) < 0.05 was considered to be significant, those < 0.01 or < 0.001 more so.