Loss of hepatic aldolase B activates Akt and promotes hepatocellular carcinogenesis by destabilizing Aldob/Akt/PP2A protein complex

Loss of hepatic fructose-1, 6-bisphosphate aldolase B (Aldob) leads to a paradoxical upregulation of glucose metabolism to favor hepatocellular carcinogenesis but the upstream signaling events remain poorly defined. Akt is highly activated in HCC and targeting Akt is being explored as a potential therapy for HCC. Herein we demonstrate that Aldob suppresses Akt activity through a protein complex containing Aldob, Akt, and protein phosphatase 2A (PP2A), leading to inhibition of cell viability, cell cycle progression, glucose metabolism and tumor growth. Interestingly, Aldob directly interacts with phosphorylated Akt (p-Akt) and promotes the recruitment of PP2A to dephosphorylate p-Akt, and this scaffolding effect of Aldob is independent of its enzymatic activity. Loss of Aldob or disruption of Aldob/Akt interaction in Aldob R304A mutant restores Akt activity and tumor promoting effects. Consistently, Aldob and p-Akt expression are inversely correlated in human HCC tissues, and Aldob downregulation coupled with p-Akt upregulation predicts a poor prognosis for HCC. We have further discovered that a specific small-molecule activator of PP2A (SMAP) efficiently attenuates HCC tumorigenesis in Aldob-deficient cell lines and xenografts. Our work reveals a novel non-glycolytic role of Aldob in negative regulation of Akt activation, suggesting that inhibiting Akt activity and reactivating PP2A may be a potential therapeutic approach for HCC treatment.


Introduction
Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and the 4 th leading cause of cancer-related deaths worldwide [1]. Despite considerable improvement in the diagnosis and treatment for HCC, the clinical prognosis of HCC remains disappointing primarily as a result of an incomplete understanding of the molecular mechanisms underlying HCC progression and limited therapeutic options [2].
Abnormal activation of the PI3K/Akt signaling pathway is a hallmark of many human malignancies, especially HCC [3]. In response to growth factors or cytokines, activated PI3K generates phosphatidylinositol-3, 4, 5-trisphosphate (PIP3), which recruits Akt for phosphorylation at threonine 308 (T308) by phosphoinositidedependent kinase 1 (PDK1) and serine 473 (S473) by mechanistic target of rapamycin complex 2 (mTORC2) [4]. Activated Akt triggers the subsequent cellular response through the phosphorylation of various downstream substrates or induction of multiple gene expression. For instance, inhibitory phosphorylation of glycogen synthase kinase 3β (GSK3β) by active Akt leads to increased stability and accumulation of CyclinD1, thereby promoting cell cycle progression [5]. Besides, activated Akt enhances the expression and activity of numerous glycolytic enzymes, such as glucose transporter, hexokinase 1 (HK1), and phosphofructokinase 1, resulting in the upregulation of glucose uptake and glycolysis [6,7]. Importantly, a series of feedback controls counteract Akt activation to maintain the transient signal. In addition to signal termination by phosphatase and tensin homolog (PTEN), protein phosphatase 2A (PP2A) and PH domain and leucine rich repeat protein phosphatase (PHLPP) function as Akt phosphatases directly dephosphorylating Akt [8,9]. Therefore, a delicate 4 balance between protein kinase-catalyzed phosphorylation and protein phosphatasemediated dephosphorylation is vital for Akt kinase activity in cellular homeostasis; dysregulation of this balance may lead to tumorigenesis. However, the upstream signaling networks involved in the regulation of Akt activity remain to be fully characterized.
Fructose-1, 6-bisphosphate aldolase catalyzes the cleavage of fructose-1, 6bisphosphate (FBP) to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate in glycolysis. Aldolase B (Aldob) is abundantly expressed in the liver, kidney and small intestine, whereas aldolase A (Aldoa) and aldolase C (Aldoc) are the muscle isoform and central nervous system isoform, respectively [10]. In humans, gene dysfunction or deficiency of Aldob causes hereditary fructose intolerance (HFI), a recessively inherited disorder of fructose metabolism [11]. Aldolase has been implicated in diverse physiological and pathological processes [12]. A recent study has identified that aldolase acts as a sensor of FBP and glucose availability in the regulation of AMPactivated protein kinase (AMPK) [13]. Dissociation of aldolase from the actin cytoskeleton leads to increased aldolase activity and enhanced glycolytic flux, which is positively regulated by PI3K signaling [14]. Aldob has been documented to be downregulated in HCC tissues in transcriptomic and proteomic studies, which is correlated with multiple malignant characteristics of HCC [15][16][17]. Our recent study identified a novel tumor-suppressive mechanism by which Aldob regulates metabolic reprogramming in HCC by interacting with the rate-limiting enzyme in pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD). Loss of Aldob leads 5 to a novel mode of metabolic reprogramming in favor of glucose metabolism by upregulating glycolysis, PPP and TCA for HCC progression. However, the upstream signaling events leading to HCC progression and metabolic reprogramming due to the loss of Aldob remain largely unknown.
Here we report that Aldob negatively regulates Akt activation, which is required for Aldob-induced suppression of cancer cell proliferation, glucose metabolism and tumorigenesis. Aldob directly binds to p-Akt potentiating PP2A interaction and dephosphorylation of p-Akt, resulting in the inhibition of Akt phosphorylation and downstream oncogenic signaling. This novel protein interaction appears to be independent of Aldob enzymatic activity. Moreover, a novel small molecule activator of PP2A (SMAP) elicits anti-tumor efficacy comparable to Akt allosteric inhibitor (MK2206) in blocking the tumorigenic effects driven by Aldob deficiency in vitro and in vivo. Interestingly, we discover an inverse correlation between Aldob and p-Akt expression in HCC tumor tissues, and a combination of low Aldob and high p-Akt expression is associated with the worst prognosis for HCC patients. Collectively, our work highlights that targeting Akt is a viable approach to treat HCC and PP2A activation using small molecule modulators of this phosphatase has the potential as a new targeted approach for HCC treatment.

Results
Aldob expression is negatively correlated with Akt activation in human HCC and the expression of Aldob/p-Akt predicts overall survival of HCC patients 6 Our recent study has revealed a novel tumor suppressive role for the glycolytic enzyme Aldob in HCC through directly binding to G6PD and inhibiting its activity, acting as a metabolic switch in glucose metabolism and regulating the metabolic reprogramming. Accumulating body of evidence has demonstrated that metabolic reprogramming in cancer cells to meet increased bioenergetic and biosynthetic requirements during tumorigenesis depends on multiple intracellular signaling pathways [18]. Among them, activated Akt signaling has a profound impact on metabolic reprogramming through upregulating glycolytic enzymes and promoting aerobic glycolysis [19]. Therefore, we set out to examine the role of Akt signaling in HCC in the context of Aldob downregulation and metabolic reprogramming. We first investigated the clinical relevance between Aldob expression and Akt activation in human HCC. As showed in Fig 1A, reduced Aldob expression and enhanced Akt phosphorylation levels at both the threonine 308 (pT308-Akt) and serine 473 (pS473-Akt) residues were observed in tumor tissues as compared to matched adjacent normal liver tissues. Next, we performed tissue microarray (TMA) analysis in paired clinical samples from HCC patients (Fig 1B; S1 Table, n=70). Aldob expression was negatively correlated with Akt activation in human HCC tissues (Fig 1C, R 2 =0.146, p=0.001). We also found that low Aldob expression was significantly correlated with α-fetoprotein (AFP), albumin level, and tumor encapsulation (S1 Table). Kaplan-Meier survival analysis showed that the overall survival time for patients with low Aldob expression was notably shorter than those with high Aldob expression (Fig 1D, p=0.016).
Conversely, patients with high pT308-Akt level exhibited shorter overall survival 7 compared with patients with low pT308-Akt level (Fig 1E, p=0.029). In addition, an elevated level of p-Akt was significantly correlated with prothrombin time, number of tumors, and tumor encapsulation (S1 Table). More importantly, a low Aldob expression coupled with high pT308-Akt levels was associated with the worst overall prognosis (Fig 1F, p=0.001). In conclusion, our study uncovers a novel inverse correlation between Aldob and p-Akt expression in human HCC and low Aldob expression with high p-Akt predicts the worst prognosis for HCC patients, suggesting an important role of Akt signaling in HCC in the context of Aldob downregulation.

Aldob inhibits Akt kinase activity and downstream signaling in HCC
To investigate the relationship of Aldob downregulation and activation of Akt signaling, we observed that, in liver-specific ALDOB knockout (KO) mouse primary hepatocytes (PHs), Aldob deletion significantly augmented Akt activation compared to wild-type (WT) PHs, as evidenced by increased levels of pT308-Akt, pS473-Akt and Akt downstream targets, including pS9-GSK3β and pT389-S6K, without affecting the expression of Akt, GSK3β, and S6K (Fig 2A). Notably, we also detected a significantly enhanced expression of HK1, a key enzyme in the first irreversible step of glycolysis by catalyzing the ATP-dependent phosphorylation of glucose to glucose-6-phosphate, which plays a critical role in cellular glucose uptake and utilization [20], suggesting that loss of Aldob may facilitate glucose metabolism through activation of Akt signaling (Fig 2A). Similarly, we observed a prominent elevation of Akt kinase activity in liver tissues of ALDOB KO mice compared to WT mice after diethylnitrosamine 8 (DEN) treatment ( Fig 2B). These data suggest a potential tumor-suppressive role of Aldob through inhibiting oncogenic Akt activity, consistent with our previous work that loss of Aldob promotes DEN-induced HCC tumorigenesis in ALDOB KO mice.
To further address the role of Aldob in the regulation of Akt phosphorylation in HCC, we generated stable Aldob-overexpressing liver cancer cell lines Huh7 and LM3, which at baseline express low levels of Aldob, and found that ectopic expression of Aldob resulted in a marked reduction of Akt, GSK3β and S6K phosphorylation levels ( Fig   2C). Conversely, silencing Aldob by siRNA restored Akt phosphorylation and its downstream targets ( Fig 2D). Next, we analyzed the kinetics of Akt activation and attenuation in response to insulin ( Fig 2E) and epidermal growth factor (EGF) (Fig 2F) stimulation. Downregulation of Aldob led to increased levels of pT308-Akt, pS473-Akt with coordinate changes in immediate Akt downstream substrate pS9-GSK3β expression in a time-dependent manner (Fig 2E and 2F). Additionally, we performed an in vitro Akt kinase assay and demonstrated that overexpressed Aldob dramatically inhibited Akt kinase activity, as assessed by the decreased phosphorylation level of Akt substrate GSK3α (Fig 2G). Taken together, all these results strongly suggest that Aldob suppresses Akt phosphorylation and its kinase activity in HCC.

Aldob inhibits Akt kinase activity and suppresses HCC through impeding cell cycle progression and attenuating glycolysis and TCA metabolism
Given that aberrant activation of Akt signaling promotes cancer progression through regulating cell proliferation, metabolism and survival [3], we next investigated the 9 functional consequence of Aldob-mediated suppression of Akt kinase activity. We observed that enforced expression of Aldob inhibited cell viability and colony formation capability. Consistently, inhibition of Akt activity by MK2206, a highly selective allosteric Akt inhibitor [21], significantly blocked cell proliferation and eliminated the growth inhibitory effects of Aldob expression (Fig 3A and B). Metabolic reprogramming has been recognized as the core hallmark for various cancers [22]. Enhanced aerobic glycolysis, known as the Warburg effect, is frequently observed along with abnormally high rates of glucose uptake in rapidly proliferating cancer cells [23]. As the central route of oxidative phosphorylation, the tricarboxylic acid (TCA) cycle is also upregulated to meet increased cellular energy, biosynthesis and redox needs [24,25]. The downstream effectors of Akt signaling play a central role in cancer cell metabolic reprogramming. To further determine whether Aldob-mediated inhibition of Akt activity is involved in Aldob-induced metabolic reprogramming, we used stable isotope labeled [U-13 C6] glucose as a tracer to track intracellular metabolic flux ( Fig 3E). The M+3 ( 13 C labeled at all three positions) fraction of enriched labeled metabolites in glycolysis including pyruvate, lactate and alanine were significantly decreased in Huh7-Aldob cells compared with control ( Fig 3F). Meanwhile, Aldob overexpression also observably reduced the M+2 ( 13 C labeled at two positions) fraction of labeled metabolites in TCA cycle, such as fumarate, malate, aspartate, glutamate and citrate ( Fig 3G). Besides, blocking Akt activity by MK2206 efficiently weakening Hepatic Aldob is indispensable for glucose and fructose metabolism, and its deficiency as result of ALDOB gene mutation leads to HFI in humans [26]. The residues at Arg43, Arg46, Lys108, Lys147, Arg149, Lys 230 and Arg304 line within the active site pocket of Aldob, and play an important role in substrate binding and catalysis [27,28]. Mutations involved in aforementioned residues have been identified in the ALDOB gene of HFI patients [29,30]. To determine whether the enzymatic activity of Aldob is required for the this novel interaction, we individually mutated the seven positively charged amino acid residues to alanine and found that among the four point mutants (R43A, K108A, K230A, and R304A) with considerably reduced Aldob-Akt interaction, 13 they exhibited varied Aldob enzymatic activities: R43A and K230A (completely dead), K108A (32.1%) and R304A (49.1%) of WT Aldob (Fig 4E). Among them, R304A mutant has the most significantly attenuated interactions with Akt. Moreover, the other three mutants (R46A, K147A, and R149A) with moderately attenuated Aldob-Akt interaction, also had variable enzyme activities: R46A maintained 60.2% activity and the other two did not have significant enzyme activity ( Fig 4E). Together, these data clearly indicate that the interaction of Aldob with Akt is independent of Aldob enzymatic activity.
To further identify the Akt interacting region on Aldob, a full-length and two truncated mutants of Aldob including a.a. 1-155 (amino acid 1-155 fragment containing clone), and a.a. 1-240 were constructed to examine their Akt-binding capability.
Intriguingly, both Aldob truncated mutants failed to interact with Akt compared to the full-length Aldob, indicating that the C-terminal region of Aldob (a.a. 241-364) is most likely the region for Akt interaction (S4B Fig), consistent with the observation that R304A has the weakest Aldob-Akt interaction. We next tested whether Aldob-Akt interaction is essential for Aldob regulation of Akt activity and HCC cell growth.
Overexpression of WT Aldob significantly downregulated Akt signaling, whereas disruption of Akt/Aldob interaction in R304A mutant abrogated Aldob-induced inhibition of Akt signaling ( Fig 4F). Consistently, R304A mutant efficiently rescued cell viability, colony formation, and cell cycle progression in Aldob-overexpressing cells (Fig 4G-I). Together, these data indicate that Aldob directly interacts with Akt to suppress Akt activity and disruption of this interaction releases Akt kinase activity to 14 promote cancer cell growth.

Aldob preferentially associates with Akt in a phosphorylation-dependent manner
Because the state of Akt phosphorylation is critical for the integration of various extracellular cues to downstream signaling events for multiple biological processes, we next studied whether the phosphorylation state of Akt played any role in the regulation of the protein-protein interaction between Akt and Aldob. To this end, we examined the effect of Akt1 phospho-deficient mutants (Akt1-T308A and Akt1-S473A) on Aldobbinding capability. Compared with Akt1-WT, both Akt1 phospho-deficient mutants displayed reduced binding with Aldob, suggesting that Akt1 kinase activity is critical for Akt1 and Aldob interaction ( Fig 5A). Next, we treated Aldob and Akt1 transfected Huh7 cells with insulin or EGF to further examine the correlation between Akt1 phosphorylation status and Aldob interaction with Akt1. Interestingly, the interaction of Aldob and Akt1 tracked with the kinetics of Akt1 activation and attenuation in response to either insulin or EGF, with the binding between Aldob and Akt1 almost peaking when the levels of phosphorylated Akt (p-Akt) were the highest (Fig 5B and C). Furthermore, blocking Akt phosphorylation through MK2206 treatment prominently decreased Aldob interaction with Akt1 ( Fig 5D). Together, these results indicate that Aldob preferentially binds to the phosphorylated form of Akt.
Aldob promotes Akt interaction with PP2A and accelerates PP2A-mediated Akt dephosphorylation 15 Given that Akt interacted with Aldob in an Akt phosphorylation-dependent manner, we hypothesized that phosphatases of Akt may be involved in Aldob-induced repression of Akt activity. Interestingly, as shown in Fig 2, Akt phosphorylation at both T308 and S473 was significantly reduced in response to Aldob expression. Previous study suggested that PP2A directly dephosphorylates Akt at both T308 and S473 [31,32]. Moreover, phosphorylation of Akt at T308 has been identified to play a more crucial role in activating Akt [33,34]. Thus we examined the possible involvement of the serine/threonine phosphatase PP2A in Aldob-mediated regulation of Akt activity.
Strikingly, we observed an abundance of the catalytic subunit of PP2A (PP2AC), but little PHLPP, in the exogenous Aldob immunocomplex (Fig 6A). Meanwhile, no interaction of Aldob with the Akt kinase PDK1 was observed (Fig 6B).
Since PP2A has been shown to bind directly to Akt and dephosphorylate Akt [35], we speculated that Aldob might nucleate the Akt interaction with PP2A to trigger Akt dephosphorylation resulting in inhibition of downstream signaling. To support this hypothesis, we performed GST pull down assay and found that WT Aldob but not the Akt1-binding deficient Aldob-R304A mutant significantly enhanced Akt1 interaction with PP2AC (Fig 6C). Similarly, endogenous Akt1 pulled down both Aldob and PP2AC in the liver tissue of WT mice, whereas loss of Aldob drastically diminished Akt1immunoprecipitated Aldob and PP2AC in ALDOB KO mice (Fig 4A), further suggesting that Aldob acts as a scaffold protein for the Akt/PP2A complex. Next, we performed in vitro PP2A phosphatase activity assay using phosphorylated Akt as a substrate and observed a time-dependent decrease of p-Akt at both T308 and S473 16 residues upon incubation with active PP2A. More importantly, WT Aldob remarkably augmented the PP2A-induced reduction of p-Akt expression, while no significant effect was found on the Akt1-binding deficient Aldob-R304A mutant (Fig 6D). Furthermore, we examined the effect of okadaic acid (OA) on these processes. OA is an inhibitor of serine/threonine protein phosphatase PP1 and PP2A, exhibiting greater selectivity toward the latter up to concentration of 100 nM [36]. As shown in Fig 6E, OA treatment effectively restored Akt phosphorylation levels in Aldob-overexpressing cells, which almost comparable to untreated control cells, suggesting that Aldob-induced inhibition of Akt activity is dependent on PP2A. To further verify these results in human HCC samples, we randomly selected two pairs of HCC tissue samples for IP assays. The endogenous interaction between Aldob and Akt1 was identified in peripheral normal liver tissues of HCC patients ( Fig 6F). However, downregulated Aldob in tumor tissues significantly decreased Akt1-immunoprecipitated Aldob and PP2AC when compared to corresponding adjacent normal tissues (Fig 6F), further suggesting that Aldob/Akt/PP2A signaling is involved in human HCC progression. Taken together, Aldob interacts with p-Akt to promote the recruitment of the phosphatase PP2A and accelerate PP2A-mediated dephosphorylation, which is primarily responsible for Aldob-induced downregulation of Akt phosphorylation.

PP2A activation using SMAP downregulates Akt activity and suppresses HCC
progression PP2A is generally considered as a tumor suppressor that dephosphorylates multiple critical oncogenic proteins, such as Akt, ERK, and MYC [37]. Functional inactivation of PP2A has been linked to tumor development in many cancers [38]. Thus, pharmacologic restoration of PP2A phosphatase activity has emerged as an attractive strategy for cancer therapy [39]. Recent studies have reported that a series of specific small-molecule activators of PP2A (SMAPs) reengineered from tricyclic neuroleptics effectively activate PP2A, resulting in the dephosphorylation of key targets Akt and ERK, and blocking tumor growth of lung, prostate cancer and pancreatic neuroendocrine tumors both in vitro and in vivo [40][41][42][43]. The validation of their target specificity, mechanism of action, and pharmaceutic properties have been extensively documented [40][41][42][43]. To evaluate the utility of PP2A activation toward Aldob-mediated suppression of Akt activity, Huh7-Vector and Huh7-Aldob cells were treated with 10 μM SMAP for further functional studies. We found that Aldob overexpression and SMAP treatment alone both inhibited cell proliferation, and the combination of the two showed an additive effect on repressing cell proliferation, as measured by cell viability and clonogenic assays (Fig 7A and B). Additionally, cell cycle analysis revealed that SMAP treatment resulted in a significant increase in the G1 phase with a coordinate loss of the S population, and exacerbated Aldob-induced G1-to-S phase cell cycle arrest (Fig 7C).
Western blot analysis showed Aldob overexpression had no effect on ERK phosphorylation in the presence of either DMSO or SMAP, whereas Aldob-induced downregulation of Akt/GSK3β/CyclinD1 signaling was potentiated by SMAPmediated PP2A activation ( Fig 7D). Notably, relative to Huh7-Aldob cells that contain diminished Akt activity, Huh7-Vector cells were more sensitive to SMAP treatment.
Together, these results indicate that PP2A plays an essential role in Aldob-induced growth inhibition, and that small molecule mediated PP2A activation is beneficial to Aldob-mediated suppression of Akt signaling and HCC cell growth.
We further investigated the efficacy of SMAP on HCC tumor progression in vivo.
SMAP treatment alone resulted in dramatic inhibition of tumor growth, which is similar to Aldob overexpression-induced growth-inhibitory effects (Fig 7E-G). There was a moderate reduction in tumor burden of Huh7-Aldob tumors compared with Huh7-Vector tumors with SMAP treatment (Fig 7E-G). It is noteworthy that there was no More importantly, upregulated Aldob-induced attenuation of Akt signaling and tumor growth was greatly enhanced in response to SMAP treatment (Fig 7E-G; S5D and E   Fig). Collectively, these results support the potential therapeutic application of PP2A phosphatase reactivation and Akt kinase inhibition for the treatment of HCC with Aldob deficiency.

Discussion
Emerging studies have documented hepatic Aldob deficiency in HCC but the underlying mechanisms remain poorly defined. Our recent study uncovered a mechanism by which loss of Aldob led to a novel model of metabolic reprogramming through upregulation of glycolysis, PPP and TCA to promote HCC. This tumor promoting effect due to the loss of Aldob is achieved by releasing the inhibition on G6PD and PPP metabolism as a result of destabilizing Aldob/G6PD/p53 protein complex. However, the upstream signaling events are still unknown. In this study, we observe another non-enzymatic tumor-suppressive role of Aldob through a direct interaction with Akt in HCC, independent of Aldob enzymatic activity (Fig 8). Aldob  [44]. Here, we found that Aldob directly interacted with Akt resulting in reduced Akt phosphorylation. Although the kinase activity of Akt is critical for its interaction with Aldob, the enzymatic activity of Aldob plays a less important role in this regard. Interestingly, among these seven single mutants of Aldob, the mutant R304A significantly destroyed this interaction with Akt, leading to the restoration of Akt phosphorylation and cell growth. The active site residue R304 plays a crucial role in aldolase enzymatic mechanism and inactivating mutant at R304 residue causes HFI [45]. Although there is currently no human data linking HFI to HCC, our results warrant 21 future study on incidence of HCC in HFI populations, especially those with this mutation. On the other hand, three Akt isoforms (Akt1-3) exhibit over 80% identity at amino acid sequence, but are not functionally redundant based on different phenotypes observed by genetic deletion of each isoform in mice [3]. Akt1 played a crucial role in cell survival [46], while Akt2 maintained glucose homeostasis [47]. We found ALDOB KO significantly increased Akt2 phosphorylation at S474 site, and Aldob interacts with Akt2 in HCC cells, suggesting Aldob plays a similar role on regulating both Akt1 and Akt2 activity. Ongoing studies will examine the role of Aldob and its modulation of Akt isoform-specific signaling networks and biological outcomes.
Emerging studies have unveiled a pleotropic role of Aldolase in diverse physiological and pathological processes, in addition to the well-established role of glycolytic enzyme [12]. Our current work clearly demonstrates the great significance of Aldob-induced repression of Akt activity for the tumor-suppressive function of Aldob in HCC.
Moreover, the inverse expression patterns of Aldob and p-Akt suggest that Aldob may be applied as a potential biomarker for HCC treatment with targeted Akt therapeutics.
Our previous study identified a novel mechanism by which Aldob interacts with G6PD to reprogram metabolism in HCC. Here, we also highlight the role of Aldob in regulating glucose metabolism through inhibiting Akt activity. Extensive evidence indicates that activated Akt promotes glucose uptake and glycolysis through activating transcription factors and upregulating many glycolytic enzymes [18]. Akt increases the expression and activity of HK1 by activating HIF1α through stimulation of mTORC1 [48,49]. Indeed, our studies showed that loss of Aldob upregulates entire central carbon 22 metabolism, including glycolysis, TCA and PPP, which was accompanied by activated Akt and increased HK1 expression. Moreover, Aldob-induced inhibition of Akt activity not only reduced glucose metabolic flux to glycolysis and TCA cycle, but also induced cell cycle arrest through downregulating Akt/GSK3β/CyclinD1 signaling, thereby leading to the impairment of cell proliferation. Thus these findings fully demonstrate the important tumor-suppressive role of Aldob through modulating Akt activity and its downstream signaling events.
Our current study has identified a close functional relationship among Aldob, Akt and PP2A, providing new mechanistic insights into the crucial upstream regulators involved in the Akt phosphorylation homeostasis mediated by the coordinate regulation by protein kinases and phosphatases [50]. As a tumor suppressor, PP2A negatively regulates several oncogenic signaling pathways and controls various cellular functions, such as cell growth, cell cycle and apoptosis [51]. PP2A-B55α has been shown to dephosphorylate Akt through a direct association and negatively regulate Akt-induced cell proliferation and survival [8]. Herein we demonstrated that Aldob was involved in PP2A-induced inhibition of Akt phosphorylation at both T308 and S473 sites. Indeed, we showed that downregulated Aldob attenuated PP2A interaction and dephosphorylation of Akt, which was consistent with the inverse relationship between Aldob and p-Akt expression in tumor tissues of human HCC and ALDOB KO mice.
Furthermore, inhibition of PP2A restored Akt phosphorylation in Aldob-expressing cells, while PP2A reactivation using SMAP enhanced Aldob-induced inhibition of Akt phosphorylation. Moreover, alteration of Aldob expression had no effect on PP2AC 23 expression, and endogenous PP2AC was identified in Aldob immunocomplex. It is conceivable that Aldob interacts with p-Akt and functions as a scaffold protein to further recruit the serine/threonine phosphatase PP2A to its direct substrate Akt resulting in the inhibition of downstream Akt signaling.
Targeting key components of PI3K/Akt pathway is being explored as a therapeutic approach for cancer treatment. Several Akt small molecular inhibitors have entered different stages of clinical trials [3]. MK2206 is a potent and orally bioavailable allosteric inhibitor of Akt that specifically targets Akt inactive conformation and blocks PDK1-mediated phosphorylation and activation [21]. To date, MK2206 has shown moderate anti-tumor efficacy in vitro and in early clinical studies as a monotherapy, and exerts significantly more promising tumor inhibitory activities in combination with other agents, such as HCC first-line treatment sorafenib [52,53]. Our data demonstrated that MK2206 treatment potently inhibited Akt phosphorylation and tumorigenic effects resulted from low Aldob level. Furthermore, tumor cells with low Aldob level were more responsive to Akt inhibition than those with high Aldob expression in which Akt signaling at baseline was already reduced. Collectively, our work indicates that targeting hyperactive Akt due to the loss of Aldob in HCC may be a viable therapeutic strategy.
In addition to direct inhibition of oncogenic kinases, stimulation of endogenous phosphatases to indirectly inactivate kinase signaling has been considered as a promising anticancer therapeutic approach. PP2A is functionally inactivated in many cancers, as a result of various mechanisms including somatic mutation, increased 24 expression of endogenous PP2A inhibitors and post-translational modifications of the catalytic subunit [39]. Aberrant transcripts of tumor suppressor gene PPP2R1B were detected in 29% of HCC tumors, which associated with the development of HCC [54].
Overall, the activation of PP2A has the potential to exert anti-tumor effect toward multiple oncogenic signaling proteins that drive cancer progression. FTY720 (fingolimod) has been reported to indirectly activate PP2A through inhibiting the endogenous PP2A inhibitor SET and exhibits high anti-tumor efficacy against HCC [38,55]. In this study, we report that an orally bioavailable PP2A activator SMAP had anti-HCC activity in vitro and in vivo through its ability to simultaneously inhibit PP2A substrates Akt and ERK activity resulting in cell cycle arrest and ultimately cell death. In summary, our study uncovers the biological importance of Aldob-mediated Akt inhibition through the formation of Aldob/Akt/PP2A protein complex, independent of Aldob enzymatic activity. Our study suggests that targeting hyperactive Akt resulting from Aldob deficiency through directly inhibiting Akt kinase or reactivating PP2A phosphatase activity may serve as an anti-tumor treatment for HCC.

Mouse Models
For subcutaneous xenograft growth model, Huh7-Aldob or Vector cells (2 × 10 6 ) mixed in serum-free medium containing 50% Matrigel (BD Biosciences) were subcutaneously injected into the left and right flanks of 5-week-old male BALB/C nude mice. After 10 days, the mice were randomly assigned to control solvent and agent Cell culture and HCC tissue samples 26 The human HCC cell lines Huh7 and HCC-LM3 were purchased from Cell Bank of CAS. Primary hepatocytes were isolated from the livers of male liver-specific ALDOBknockout and WT mice using collagenase perfusion as previously described [56]. HCC

Cell viability assay
The viability assay was performed by seeding cells in a 96-well plate at a density of 2,000 cells/well for 6 repeats. After 7 hours, cell media were replaced with fresh media with various concentrations of test agents or control DMSO and cells were proceeded to grow. CCK8 (OBIO Cell Counting Kit) then was added to separate plates at the indicated time according to the manufacturer's instruction.

Colony formation assay
Colony formation assays were adapted from a protocol reported previously [33].
Cells of different groups were seeded (5000 cells/well) into six-well plates and cultured A Shimadzu QP-2010 Ultra GC-MS was programmed as described previously [57] with an injection temperature of 250°C. 1 μl of sample was injected with an injection split ratio of 1/10. GC oven temperature started at 110°C for 4 min, rising to 230°C at 3°C/min and to 280°C at 20°C /min with a final holding at this temperature for 2 min.

Data Availability Statement
All relevant data are within the paper and its Supporting Information files.

Funding
This work was financially supported by National Key R&D Program of China

Competing interests
The