Malic enzymes in cancer: Regulatory mechanisms, functions, and therapeutic implications

Abstract

Malic enzymes (MEs) are metabolic enzymes that catalyze the oxidation of malate to pyruvate and NAD(P)H. While researchers have well established the physiological metabolic roles of MEs in organisms, recent research has revealed a link between MEs and carcinogenesis. This review collates evidence of the molecular mechanisms by which MEs promote cancer occurrence, including transcriptional regulation, post-transcriptional regulation, post-translational protein modifications, and protein-protein interactions. Additionally, we highlight the roles of MEs in reprogramming energy metabolism, suppressing senescence, and modulating the tumor immune microenvironment. We also discuss the involvement of these enzymes in mediating tumor resistance and how the development of novel small-molecule inhibitors targeting MEs might be a good therapeutic approach. Insights through this review are expected to provide a comprehensive understanding of the intricate relationship between MEs and cancer, while facilitating future research on the potential therapeutic applications of targeting MEs in cancer management.

Graphical abstract

1. Introduction

In 1947, Kornberg et al. discovered an enzyme in pigeon liver that could convert malate to pyruvate using manganese ions (Mn²⁺). Subsequently, this enzyme was named malic enzyme (ME) [1]. As detection techniques advanced, scientists revealed the widespread distribution of MEs and their roles in animals, plants, and microorganisms [[2], [3], [4], [5], [6], [7], [8], [9]]. Mammalian MEs are classified into three subtypes based on differences in subcellular localization and auxiliary factors: cytoplasmic NADP⁺-dependent ME1, mitochondrial NAD(P)⁺-dependent ME2, and mitochondrial NADP⁺-dependent ME3 [10,11]. In the conversion of malate to pyruvate, MEs reduce NAD(P)⁺ to NAD(P)H. Pyruvate serves as a feeder molecule to initiate the metabolism of major nutrients, whereas NAD(P)H neutralizes reactive oxygen species (ROS) and is involved in lipid synthesis [[12], [13], [14], [15]]. MEs play crucial roles in regulating the redox balance, molecular synthesis, and energy metabolism in organisms [[16], [17], [18], [19]].

The link between MEs and cancer has garnered considerable interest recently. Dysregulation of MEs has been implicated in several types of cancers, highlighting their potential as important prognostic biomarkers for anticancer treatment outcomes [[20], [21], [22], [23]]. Despite being a group of metabolic enzymes, MEs not only promote the metabolic reprogramming of tumor cells but also contribute to ferroptosis, senescence, and alterations in the tumor immune microenvironment [[24], [25], [26]]. We summarize novel regulatory mechanisms of MEs in cancer, provide an overview of recent findings on how MEs mediate tumor resistance, and highlight the current status of ME inhibitors in cancer management. This review will provide novel insights into new targets and research strategies for the diagnosis and treatment of cancer.

2. Subcellular localization and structure of MEs

Three subtypes of MEs have been identified in mammals, namely ME1, ME2, and ME3, each exhibiting distinct subcellular localization and biological functions [10,11,27] (Fig. 1A). ME1 is an NADP⁺-dependent metabolic enzyme that primarily localizes to the cytoplasm and participates in cytoplasmic NADPH generation and fatty acid metabolism [[27], [28], [29], [30], [31]]. ME2 is the most abundant and active subtype of MEs, predominantly present in the mitochondria. ME2 regulates cellular energy metabolism by modulating mitochondrial function, the tricarboxylic acid cycle, and glutamine catabolism [22,32,33]. The conventional mitochondrial ME2 lacks an N-terminal chain of 18 amino acids, whereas the cytoplasmic full-length ME2 has this amino acid chain in its structure. Therefore, ME2 can exist in two distinct structural forms and can be found in both the cytoplasm and mitochondria [34]. ME3 is predominantly present in the mitochondria and catalyzes the oxidative decarboxylation of malate to pyruvate in the tricarboxylic acid cycle. Moreover, ME3 plays a crucial role in NADPH synthesis and maintains intracellular homeostasis in ROS levels [11,35].

Fig. 1.

Subcellular localization and structure of MEs

(A) The three subtypes of MEs exhibit distinct subcellular localization. ME1 is predominantly present in the cytoplasm; ME2 can be localized in both the cytoplasm and mitochondria; ME3 is predominantly present in the mitochondria. (B) ME monomers are composed of 572–604 amino acids, consisting of four domains (A, B, C, and D), with domain B further divided into two peptide fragments (B1 and B2).

Malic enzymes are homotetrameric proteins with a dimer-of-dimers structure, and each monomer comprises 572 to 604 amino acids. ME monomers have a molecular weight of 64–68 kDa, comprising four domains (A, B, C, and D), with domain B further divided into two peptide fragments (B1 and B2) (Fig. 1B). The formation of tetramers is facilitated primarily by domains A and D, whereas domains B and C, along with some residues in domain A, play a crucial role in enzyme catalysis [36,37]. The active site of MEs lies within a deep cleft between domains B and C, with highly conserved amino acid residues in the active site region that facilitate substrate binding, divalent cation coordination, NAD(P)⁺ cofactor binding, and enzyme catalysis regulation [10,37,38].

3. Regulatory mechanisms of MEs in cancer

We outline four key regulatory mechanisms of MEs in cancer: interaction with transcription factors, modulation by miRNAs affecting mRNA levels, involvement in various post-translational modifications influencing cancer progression, and direct protein-protein interactions altering metabolic pathways and proliferation capabilities (Fig. 2).

Fig. 2.

Regulatory mechanisms of MEs in cancer

(A) Interaction of MEs with transcription factors. MEs interact with transcription factors, such as MYC and p53, forming interaction mechanisms, or are directly activated by transcription factors such as NRF2 and ETV4, thereby mediating subsequent functions. (B) MicroRNAs negatively regulate MEs. The mRNA levels of MEs are regulated by miRNAs, such as miR-30a and miR-885-5p, thereby affecting tumor cell proliferation. (C) Post-translational modifications of MEs. MEs are involved in various post-transcriptional modifications such as phosphorylation, acetylation, methylation, and desuccinylation. (D) Interactions between MEs and proteins. MEs directly bind to proteins or form complexes, linking multiple metabolic pathways and affecting tumor progression.

Abbreviations: 6PGD: 6-phosphogluconate dehydrogenase; ACAT1: acetyl-CoA acetyltransferase 1; AKT1: AKT serine/threonine kinase 1; AML: acute myeloid leukemia; AMPK: AMP-activated protein kinase; CRC: colorectal cancer; ETV4: ETS variant transcription factor 4; GC: gastric cancer; HCC: hepatocellular carcinoma; HTC: hydride transfer complex; LKB1: liver kinase B1; LSCC: laryngeal squamous cell carcinoma; MDH1: malate dehydrogenase 1; MDM2: murine double minute 2; ME1: malic enzyme 1; ME2: malic enzyme 2; ME3: malic enzyme 3; mTORC1: mammalian target of rapamycin complex 1; MYC: MYC proto-oncogene, bHLH transcription factor; NEK1: NIMA-related kinase 1; NRF2: nuclear factor erythroid 2-related factor 2; OS: osteosarcoma; OV: ovarian cancer; PC: pyruvate carboxylase; PGAM5: PGAM family member 5; PRAD: prostate adenocarcinoma; PRMT1: protein arginine methyltransferase 1; ROS: reactive oxygen species; SIRT5: sirtuin 5; TCL: T cell lymphoma; TF: transcription factor.

3.1. Interaction of MEs with transcription factors

Transcription factors target MEs based on the cellular context, meticulously adjusting their activity to address diverse environmental and physiological challenges. This precise regulatory mechanism is crucial for optimizing cellular metabolism to suit specific needs, thereby influencing cancer progression and treatment outcomes [21,22,35,43]. These regulatory factors are essential in human physiology and disease management, as they modulate gene expression by recognizing specific DNA sequences, significantly affecting various cancer phenotypes and functions [[44], [45], [46]].

In certain tumors, specific transcription factors activate MEs and speed up cancer progression [21,43] (Fig. 2A). For example, in hepatocellular carcinoma, nuclear factor erythroid 2-related factor 2 (NRF2) is activated by extracellular oxidative stress and subsequently enhances the transcription of ME1. This mechanism is tailored to mitigate ROS damage and bolster the antioxidant defenses in hepatocellular carcinoma cells, which inadvertently supports their proliferation [21]. In gastric cancer, ME1 is upregulated by ETS variant transcription factor 4 (ETV4) transcription, leading to increased resistance to apoptosis and enhanced proliferation of gastric cancer cells. Moreover, ME1 promotes tumor growth and metastasis of gastric cancer in vivo [43].

MEs are also regulated by multiple transcriptional regulatory factors through feedback loop mechanisms, thereby participating in tumorigenesis and progression [22,35] (Fig. 2A). For instance, in a mouse model with both ME2 gene knockout and MYC overexpression, MYC upregulated ME2 and stimulated mTORC1 activity by enhancing glutamine metabolism [22]. This promoted MYC translation, forming a positive feedback loop that maintained redox homeostasis and enhanced the proliferation of T cells in lymphoma [22]. In another study, p53, activated by DNA damage signals, inhibited the transcription of ME1 in colorectal cancer cells, and downregulation of ME1 activates p53 through the inhibition of murine double minute 2 (MDM2) expression. Simultaneously, p53 transcriptionally suppressed ME2, upregulating AMP-activated protein kinase (AMPK) expression and leading to further phosphorylation and activation of p53. In summary, ME1/ME2 inhibited by p53, in turn activates p53, ultimately enhancing the senescence phenotype and reducing the proliferation of colorectal cancer cells [35].

These studies indicate that the specific transcriptional regulatory mechanisms associated with MEs affect the redox balance, energy metabolism, and pathways such as cell senescence or apoptosis, in cancer cells and play pivotal roles in cancer onset and progression. Interventions targeting MEs or their transcriptional regulatory pathways may offer new options for treating malignant tumors.

3.2. MicroRNAs negatively regulate MEs

MicroRNAs (miRNAs) are post-transcriptional regulators that mediate mRNA degradation or translational inhibition through complementary pairing with target mRNA, thereby regulating the development of drug resistance in cancer [[47], [48], [49]]. Some miRNAs have been identified to inhibit the mRNA levels of MEs and subsequently attenuate cancer progression [[50], [51], [52]] (Fig. 2B). For example, in KRAS-mutant colorectal cancer cells, miR-30a suppressed ME1 expression by binding to its 3′ untranslated region. Downregulation of ME1 expression can reduce NADPH synthesis, lipid synthesis, and fatty acid uptake in cells, inhibiting the proliferation of colorectal cancer cells both in vitro and in vivo [50]. Another miRNA, miR-885-5p, was reported to reduce the proliferation, migration, and invasion of laryngeal squamous cell carcinoma cells by suppressing ME1 expression [52]. In gastric cancer cells, miR-885-5p inhibits the expression of ME1 and that of its downstream molecules, vimentin and fibronectin. As a result, the migration, invasion, and metastasis of gastric cancer are inhibited [51].

Current research on miRNAs and MEs has focused on the ME1 subtype, which contributes to our understanding of the involvement of MEs in cancer at the post-transcriptional level [[50], [51], [52]]. These studies have emphasized the importance of ME1 as an adverse prognostic factor in various cancers, suggesting that the downregulation of ME1 expression may hold therapeutic potential for cancer management.

3.3. Post-translational modifications of MEs

Post-translational modifications occur on specific amino acids within the regulatory domains of target proteins, increasing the functional diversity of the proteome in complex ways. Such modifications have emerged as therapeutic targets in cancer [[53], [54], [55]]. MEs can influence cancer progression after undergoing various post-translational modifications, including phosphorylation, acetylation, methylation, and desuccinylation [20,23,32,34,56] (Fig. 2C).

One such modification is phosphorylation, which is crucial for ME activity. AKT serine/threonine kinase 1 (AKT1) phosphorylates the Ser9 site of cytoplasmic ME2, enhancing its enzymatic activity and preventing its mitochondrial translocation. Cytoplasmic ME2 assembles various glycolytic enzymes, including PFKL, GAPDH, PKM2, and LDHA, ultimately facilitating the metabolic switch from mitochondrial metabolism to glycolysis, thereby resulting in tumorigenesis [34].

Acetylation is another pronounced post-translational modification. The dynamic regulation of phosphorylation and acetylation in ME1 plays a crucial role in energy metabolism and disease progression in colorectal cancer [20]. NIMA-related kinase 1 (NEK1) phosphorylated ME1 at the S336 site, inhibiting its activity and suppressing tumor growth in colorectal cancer [20]. In contrast, PGAM family member 5 (PGAM5) dephosphorylated the S336 site, allowing acetyl-CoA acetyltransferase 1 (ACAT1) to acetylate the adjacent K337 site [20]. After acetylation, ME1 underwent dimerization, which enhanced its ability to catalyze NADPH and lipid production, promoting the proliferation of colorectal cancer cells [20]. Acetylation is the process of transferring and adding acetyl groups to lysine residues or N-termini of proteins, which to some extent influences the occurrence and development of tumors [[57], [58], [59]]. The dynamic balance between phosphorylation at S336 and acetylation at K337 of ME1 suggests a potential mechanism through which metabolic enzymes compete for modifications, thereby governing tumor signaling [20]. Moreover, research on ME activity emphasizes the importance of NADPH production. In osteosarcoma cells, silencing ME1/ME2 reduced NADPH levels resulting in disrupted histone acetylation and transcription processes [56].

Another form of modification is methylation, which is closely associated with tumorigenesis [[60], [61], [62]]. Mitochondrial overdrive is a main contributor to acute myeloid leukemia development, and ME2 enhances the invasiveness of leukemia cells and promotes tumor progression in vivo [32]. Methylation of ME2 by protein arginine methyltransferase-1 (PRMT1) at the fumarate-binding site (R67) of ME2 inhibits its dimerization and activity, suppressing fumarate signaling transduction and mitosis, thereby reducing the invasiveness of acute myeloid leukemia [32].

Desuccinylation has been found to significantly impact cancer progression, as evidenced by reports that desuccinylation of ME2 influences this process [23]. Succinylation involves attaching succinyl groups to lysine residues of substrate proteins, thereby altering protein structure and function. This modification has been found to regulate tumor growth [[63], [64], [65]]. The succinylation of ME2 is considered a physiological post-translational modification in colorectal cancer cells. Protein lysine desuccinylase 5 (SIRT5) desuccinylates the K346 site of ME2, activating it and subsequently enhancing mitochondrial respiration and cell proliferation [23]. Moreover, desuccinylation of the K346 site of ME2 promotes tumor growth in vivo [23].

These data suggest that post-translational modifications of MEs regulate tumor progression by influencing mitochondrial function, NADPH synthesis, and metabolic reprogramming.

3.4. Interactions between MEs and proteins

MEs can influence tumor development by directly binding to proteins or forming complexes with other molecules [24,66,67] (Fig. 2D). For example, ME1 can directly bind to and activate 6-phosphogluconate dehydrogenase (6PGD) and promote NADPH generation, pentose phosphate pathway flux, and the proliferation of osteosarcoma cells, suggesting direct interactions between MEs and the pentose phosphate pathway [66]. In the cytoplasm of prostate cancer cells, the tricarboxylic acid cycle (TCA) involves ME1, pyruvate carboxylase (PC), and malate dehydrogenase 1 (MDH1), resulting in the formation of intermediate transfer complex (HTC). This complex provides cells with NAD⁺ and NADPH, driving metabolic reprogramming to suppress senescence and induce tumorigenesis [24]. However, MEs are not always carcinogenic. In ovarian cancer cells, liver kinase B1 (LKB1) promotes the function of ME3 by binding to its N-terminus. Upregulation of ME3 expression enhances the transcription of p21 and p53 while inhibiting the transcription of NF-κB, promoting apoptosis and inhibiting cancer cell proliferation [67]. The regulatory effect of MEs on the development of pelvic malignancies and osteosarcoma is complex, and interventions targeting these targets may provide more therapeutic opportunities for patients with cancer.

4. Biological functions of MEs in cancer

MEs are dysregulated in various types of cancers and are closely linked to cancer prognosis (Fig. 3, Table 1). MEs are upregulated in several cancers, and are usually associated with shorter overall survival. However, MEs are found to be downregulated in certain types of cancers, such as bladder cancer, which is associated with shorter overall survival duration. To provide a more detailed description of malic enzymes in these cancers and their association with overall survival, we have supplemented our analysis with additional data from the UALCAN database (presented in Fig. 3). MEs play vital roles in cancer development. Owing to the recent advancement in understanding the complex biological role of MEs in cancer, this section focuses on the impact of MEs on tumor metabolic reprogramming, programmed cell death, senescence and cell cycle progression, and the tumor immune microenvironment (Fig. 4).

Fig. 3.

Dysregulation and survival relevance of MEs in various cancer types

Triangles represent the expression levels of MEs in cancer, with red indicating upregulation, blue indicating downregulation, and no color indicating no statistically significant difference in expression levels or N/A. Circles represent the relationship between expression levels of MEs and survival outcomes. Red circles indicate that high expression predicts unfavorable survival, blue circles indicate that high expression predicts favorable survival, and circles with no color and diagonal lines indicate that expression levels are not significantly associated with survival. Content within the dashed lines is supplementary data from the UALCAN database. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Table 1.

The role of MEs in cancer.

ME	Cancer types	Expression	Effect on cancer	Prognosis	Biological functions	Ref
ME1	Lung cancer	Upregulated	Promote	/	Proliferation, metabolic reprogramming	[82]
	Lung cancer	Upregulated	Promote	Poor	Proliferation	[104]
	hepatocellular carcinoma	Upregulated	Promote	Poor	Proliferation, migration, invasion, metastasis, metabolic reprogramming	[21]
	hepatocellular carcinoma	Upregulated	Promote	/	/	[109]
	hepatocellular carcinoma	/	Promote	/	Proliferation, metabolic reprogramming	[81]
	Osteosarcoma	/	Promote	/	Proliferation, metabolic reprogramming	[66]
	Laryngeal squamous cell carcinoma	Upregulated	Promote	/	/	[52]
	Synovial sarcoma	Downregulated	/	/	Ferroptosis, metabolic reprogramming	[25]
	Colorectal cancer	/	Promote	/	Senescence, proliferation, metabolic reprogramming	[35]
	Colorectal cancer	Upregulated	Promote	/	Proliferation, metabolic reprogramming	[20]
	Colorectal cancer	Upregulated	Promote	/		[96]
	Colorectal cancer	Upregulated	Promote	/	Proliferation, metabolic reprogramming	[50]
	Colorectal cancer	Upregulated	Promote	/	Senescence, apoptosis, proliferation, metabolic reprogramming	[85]
	Oral squamous cell carcinoma	Upregulated	Promote	/	Proliferation, invasion	[102]
	Oral squamous cell carcinoma	Upregulated	Promote	/	EMT, metabolic reprogramming	[110]
	Oral squamous cell carcinoma	Upregulated	Promote	Poor	Proliferation, invasion, EMT, stemness	[111]
	Prostate cancer	Upregulated	Promote	/	Senescence, proliferation, metabolic reprogramming	[24]
	Breast cancer	/	Promote	/	Proliferation	[112]
	Breast cancer	Upregulated	Promote	Poor	Proliferation, metabolic reprogramming	[76]
	Breast cancer	/	Promote	/	Proliferation, apoptosis, metabolic reprogramming	[113]
	Gastric cancer	Upregulated	Promote	Poor	Proliferation, apoptosis, metastasis	[43]
	Gastric cancer	Upregulated	Promote	Poor	Migration, invasion, metastasis	[51]
	Ovarian cancer	Upregulated	Promote	/	Senescence, apoptosis, proliferation, metabolic reprogramming	[85]
	Acute myeloid leukemia	Upregulated	Promote	Poor	Proliferation, metabolic reprogramming	[103]
ME2	Acute myeloid leukemia	Upregulated	Promote	Poor	Proliferation, metabolic reprogramming	[32]
	Acute myeloid leukemia	Upregulated	Promote	/	Apoptosis, metabolic reprogramming	[77]
	Lymphoma	Upregulated	Promote	Poor	Proliferation, metabolic reprogramming	[22]
	Lung cancer	Upregulated	Promote	/	Migration, invasion	[27]
	Lung cancer	Upregulated	Promote	/	Proliferation, metabolic reprogramming	[34]
	hepatocellular carcinoma	Upregulated	Promote	Poor	Proliferation, cell cycle	[93]
	hepatocellular carcinoma	Upregulated	Promote	Poor	Proliferation, metabolic reprogramming	[33]
	hepatocellular carcinoma	/	Promote	/	Migration, invasion	[114]
	hepatocellular carcinoma	Upregulated	Promote	Poor	Proliferation, migration, EMT, cell cycle, apoptosis	[92]
	Melanoma	Upregulated	Promote	/	Proliferation, migration, invasion, metabolic reprogramming	[115]
	Glioblastoma	Upregulated	Promote	Poor	Proliferation, migration, invasion, PMT, metabolic reprogramming	[80]
	Colorectal cancer	/	Promote	/	Senescence, proliferation, metabolic reprogramming	[35]
	Colorectal cancer	Upregulated	Promote	Poor	Proliferation, metabolic reprogramming	[23]
	Oral squamous cell carcinoma	Upregulated	Promote	Poor		[116]
	Breast cancer	Upregulated	Promote	Poor	Metastasis	[117]
	Salivary gland cancer	Upregulated	Promote	/	Proliferation	[118]
ME3	Bladder cancer	Downregulated	Suppress	Good		[119]
	Ovarian cancer	/	Suppress	/	Proliferation, apoptosis	[67]
	Pancreatic cancer	Upregulated	Promote	/	Apoptosis, metabolic reprogramming	[16]
	Pancreatic cancer	Upregulated	Promote	Poor	Proliferation, migration, invasion, EMT	[120]
	Acute myeloid leukemia	Upregulated	Promote	Poor	Proliferation, metabolic reprogramming	[103]
	hepatocellular carcinoma	Upregulated	Promote	Poor	Proliferation, migration, invasion, metastasis, metabolic reprogramming	[21]

Fig. 4.

Biological functions of MEs in cancer

The outer circle delineates the subtypes of MEs, with ME1 denoted by a blue circle, ME2 by a purple circle, and ME3 by a yellow circle. (A) MEs in tumor metabolic reprogramming. MEs reprogram the metabolism of tumor cells by mediating aerobic glycolysis, fatty acid metabolism, and glutamine degradation. The mitochondrial activity and redox balance of tumor cells are altered as a result. (B) MEs in programmed cell death. Silencing of MEs induces apoptosis by increasing the activity of caspase-3,7 or ROS. The absence of ME1 leads to the accumulation of ROS and active iron, thereby increasing ferroptosis. (C) MEs in senescence and cell cycle progression. MEs modulate the senescence phenotype by forming complexes with other molecules. MEs play a role in regulating cell cycle progression through direct or indirect mechanisms. (D) MEs in the tumor immune microenvironment. ME1 overexpression in CD8⁺ T cells increases mitochondrial respiration rate and ATP production, contributing partially to the enhanced cytotoxicity of CD8⁺ T cells. Downregulation of ME1 sensitizes tumor cells to the toxicity of natural killer cells-derived HMGB1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

4.1. MEs in tumor metabolic reprogramming

Cancer cells have the ability regulate their energy metabolism, favoring their proliferation and differentiation. Therefore, reprogramming of energy metabolism is a hallmark of malignant cancers [[68], [69], [70]]. Typically, metabolic reprogramming in cancer occurs in pathways of aerobic glycolysis, fatty acid metabolism, and glutamine metabolism [71,72]. MEs can mediate tumor onset and progression by promoting aerobic glycolysis, fatty acid metabolism, and glutamine metabolism, as discussed in subsequent sections (Fig. 4A).

4.1.1. MEs in aerobic glycolysis

Aerobic glycolysis, also known as the Warburg effect, refers to the phenomenon in which tumor cells convert glucose to lactate even in the presence of oxygen, to satisfy their own needs for proliferation and metastasis. It is the most extensively studied phenomenon in the metabolic reprogramming of malignant cancers [[73], [74], [75]]. MEs can mediate aerobic glycolysis and influence tumor progression. For example, ME1 overexpression increases the production of lactate in breast cancer cells, which increases the extracellular acidification rate (ECAR) and decreases the oxygen consumption rate (OCR), thereby promoting aerobic glycolysis and facilitating tumor growth [76]. Downregulation of ME2 in acute myeloid leukemia decreases the expression of glycolytic intermediates, effectively inhibits glycolysis, and suppresses acute myeloid leukemia cell proliferation [77].

4.1.2. MEs in fatty acid metabolism

The metabolic reprogramming of fatty acid metabolism is reported to occur in several cancer types, which activates the biosynthesis of fatty acids to satisfy the proliferation demands of cancer cells [71,78,79]. MEs can promote tumor cell fatty acid metabolism [50,80,81]. For instance, ME2 enhances the de novo synthesis of fatty acids via the AMPK-SREBP-1-ACSS2 pathway, thereby promoting the proliferation, migration, and invasiveness of glioblastoma multiforme cells [80]. Knocking out ME1 significantly reduces triglyceride and fatty acid levels in KRAS-mutant colorectal cancer cells, thereby inhibiting their tumorigenicity [50]. PGAM5 mediates ME1 dephosphorylation and enhances ME1 activity, thereby promoting NADPH production, fatty acid metabolism, and tumor growth in hepatocellular carcinoma cells [81]. Considering that NADPH provides the essential reducing power for lipid metabolism, an increase in NADPH production through the action of ME1 has been reported to enhance the growth of various tumors [21,24,82].

4.1.3. MEs in glutamine catabolism

Glutamine is another important nutrient in addition to glucose and fats. Glutamine is broken down into various metabolites to satisfy the energy demands of tumor cells without compromising the redox balance in cancer cells [83,84]. Previous studies have indicated that silencing ME1 or ME2 can reduce glutamine breakdown in colorectal cancer cells, suggesting that MEs play crucial roles in glutamine catabolism [35]. Glutamine, rather than glucose, is the main nutrient for MYC/ME2-driven T-cell lymphoma cells, and ME2 overexpression can restore glutamine consumption in MYC-deleted cells, thereby promoting cell proliferation [22]. ME2 also maintains the levels of 2-hydroxyglutarate in glioblastoma cells by regulating glutamine breakdown. The direct binding of 2-hydroxyglutarate with mutant p53 inhibits the MDM2-mediated ubiquitination and degradation of mutant p53, thereby promoting tumor growth [33].

MEs play important roles in maintaining mitochondrial function and cellular redox balance [21,32]. For instance, in acute myeloid leukemia, ME2 can target and activate deoxyuridine triphosphate nucleotidohydrolase, elevating the mitochondrial levels of dNTP and mtDNA and consequently increasing mitochondrial biomass in acute myeloid leukemia cells [32]. In hepatocellular carcinoma, ME3 facilitates the production of a large amount of mitochondrial NADPH, which helps maintain redox balance; conversely, silencing ME3 directly reduces mitochondrial activity in hepatocellular carcinoma cells [21].

In summary, MEs accelerate tumor progression by modulating nutrient metabolism reprogramming and mitochondrial activity. Therefore, targeting MEs may offer a novel therapeutic strategy for inhibiting tumor metabolism.

4.2. MEs in programmed cell death

MEs inhibit tumor cell apoptosis through various mechanisms, promoting tumor development [43,77,85]. For instance, silencing ME1 in gastric cancer cells induces apoptosis and significantly inhibits tumor growth [43]. In non-small cell lung cancer cells, silencing ME1 significantly increases the activity of caspase-3, 7 and induces apoptosis [85]. Furthermore, ME2 downregulation leads to increased ROS, thus resulting in apoptosis of acute myeloid leukemia cells [77].

Unlike apoptosis, ferroptosis is a newly identified iron-dependent form of programmed cell death that generates large amounts of lipid peroxidation products, ultimately inducing cell death and playing a crucial role in cancer progression [[86], [87], [88]]. The absence of ME1 in synovial sarcoma cells leads to the accumulation of ROS and active iron, increasing the occurrence of ferroptosis and enhancing the sensitivity of cells to cystine transporter SLC7A11/xCT inhibitors that induce ferroptosis [25]. Loss of ME1 expression exacerbates ischemia-reperfusion-induced hepatocyte ferroptosis, whereas supplementation of the ME1 substrate l-malate restores NADPH and glutathione levels, thereby inhibiting ischemia-reperfusion-induced hepatocyte ferroptosis [17].

In summary, MEs regulate different types of programmed cell death, thereby affecting tumor progression and treatment resistance (Fig. 4B). Therefore, interventions that target MEs may, at least, partially address the challenge of treatment resistance in cancer therapy.

4.3. MEs in senescence and cell cycle progression

Senescence is a state of cell cycle arrest and a protective mechanism against tumorigenesis, with microenvironmental stress and imbalances in cellular signaling networks as triggers of senescence [[89], [90], [91]]. MEs are reported to promote tumor growth by inhibiting cancer cell senescence [24,85] (Fig. 4C). For instance, murine and human prostate cancer models exhibit overexpression of the hydrogen transfer complex (HTC), consisting of ME1, PC, and MDH1. Silencing any of these three enzymes in the HTC induces senescence and leads to the arrest of proliferation [24]. Similarly, prostate cells lacking ME1 exhibit a higher proportion of SA-β-Gal-positive cells, another hallmark of senescence, suggesting that knocking out ME1 induces senescence and inhibits cancer cell growth [85]. The mutual regulation between p53 and MEs plays a major role in regulating senescence in normal and cancer cells [35].

MEs also mediate tumor progression by regulating the cell cycle process [92,93] (Fig. 4C). ME2 promotes the binding of α-ketoglutaric acid to RNA polymerase II, indirectly increasing cyclin D1 transcription and thus promoting cell cycle progression and the proliferation of hepatocellular carcinoma cells [93]. An increase in the number of non-proliferating hepatocellular carcinoma cells in the G0 phase has been reported after silencing ME2, indicating that ME2 downregulation can inhibit cell cycle progression and proliferation [92]. These data suggest that MEs mediate tumor progression either by inhibiting senescence or by promoting cell cycle progression, highlighting that cellular metabolism is a major predictor of the irreversible fate of cells.

4.4. MEs in the tumor microenvironment

Malignant tumors are not simply a cluster of isolated cell types but rather a complex ecosystem composed of immune cells, stromal cells, blood vessels, nerves, lymphatic vessels, and other types of cells [94,95]. ME1 regulates the activity of CD8⁺ T cells and natural killer cells through different mechanisms and alters the tumor microenvironment (TME) [26,96] (Fig. 4D). For instance, salvage therapy sometimes can activate cytotoxic T cells in patients with refractory advanced cancer, leading to tumor regression [26]. Analysis of peripheral blood lymphocytes from patients with late-stage lung metastasis patients who received salvage therapy with spatially fractionated radiotherapy showed that the treatment increased the toxicity of CD8⁺ T cells by upregulating ME1. ME1 overexpression in CD8⁺ T cells significantly increased mitochondrial respiration rate and ATP production, which contributed partially to the enhanced cytotoxicity of CD8⁺ T cells through the type I interferon pathway [26]. Natural killer cells-derived high-mobility group box 1 (HMGB1) can also effectively induce cell death in colorectal cancer cells, and downregulation of ME1 sensitizes glutamine-dependent colorectal cancer cells to the toxicity of HMGB1 [96].

TME, comprised of diverse immune cell subsets, plays a critical role in tumor development and metastasis. Ongoing advancements in combined immunotherapy, built upon understanding the regulatory mechanisms within the TME, are being continuously refined [97,98]. Research exploring the link between malic enzymes and the TME provides valuable insights into combined therapies for cancer. Further investigation in this field will deepen our understanding of the underlying molecular mechanisms.

5. ME-mediated drug resistance

Drug resistance poses a major challenge that prevents us from achieving favorable outcomes despite advancements in cancer treatment strategies, and genetic and non-genetic mechanisms are reported to contribute to resistance to cancer treatment [[99], [100], [101]]. MEs can influence drug resistance in hematologic and epithelial malignancies through various pathways [21,82,102,103] (Fig. 5). For instance, in highly invasive acute myeloid leukemia, ME1/ME3 modulates cellular oxidative metabolism by balancing the levels of NADPH and ROS, thereby mediating resistance to cytarabine. Targeting ME1/ME3 effectively reduces the tumorigenicity of acute myeloid leukemia and helps overcome resistance [103]. In hepatocellular carcinoma, ME1/ME3 downregulation leads to increased apoptosis, reduced proliferation, and ROS induction after sorafenib treatment, suggesting that interference with the ME1/ME3 pathway sensitizes hepatocellular carcinoma to sorafenib therapy [21]. ME1 and G6PD serve as primary NADPH contributors in tyrosine kinase inhibitor-resistant non-small cell lung cancer cells, and the administration of ME1 inhibitors effectively overcomes cancer resistance, especially when combined with osimertinib [82]. The high expression of ME1 maintains lactate fermentation levels by promoting glutamine metabolism, enhancing proliferation, and increasing resistance in oral squamous cell carcinoma [102].

Fig. 5.

ME-mediated drug resistance

MEs influence the sensitivity of cancers originating from epithelial tissues, mesenchymal tissues, and the hematopoietic system, to chemotherapy drugs, targeting agents, and ferroptosis inducers.

Depletion of ME1 expression makes mesenchymal tissue-derived malignant tumors more sensitive to ACXT-3102, which is an erastin compound that induces ferroptosis by inhibiting xCT-mediated cystine uptake [25]. Depletion of ME1 expression also alters the antioxidant defense system in synovial sarcoma cells, leading to the accumulation of ROS and labile iron, thereby increasing the sensitivity of these cells to ACXT-3102-induced ferroptosis. This further supports the development of therapeutic strategies targeting ferroptosis pathways in synovial sarcoma [25]. Moreover, patients with non-small cell lung cancer who exhibit increased ME1 expression typically experience poorer clinical outcomes following radiotherapy. This relationship underscores the potential of targeting ME1 to enhance radiotherapy efficacy, offering a promising direction for future therapeutic strategies [104].

In summary, MEs mediate tumor resistance by influencing cellular redox balance, glutamine metabolism, NADPH synthesis, and other processes in malignant tumors of the hematopoietic system and epithelial tissues. Conversely, in tumors originating from mesenchymal tissues, MEs affect tumor resistance by modulating the sensitivity of these cancer cells to ferroptosis induction. This highlights the potential of MEs as emerging biological markers in cancer therapy and provides new directions and insights for future research endeavors.

6. ME inhibitors

The various subtypes of human MEs share a similar quaternary structure, forming a homotetrameric protein (Fig. 6A). ME2 has a complex allosteric system, with each enzyme monomer containing two independent allosteric sites: the fumarate site at the dimer interface and an exosite at the tetramer interface. ME2 can shift between two open and two closed forms in equilibrium [10,[39], [40], [41], [42]]. ME1 and ME3 are non-allosteric enzymes with relatively stable tetrameric structures [37,38]. From the first identification of the crystal structure of human ME in 1999 to the elucidation of the crystal structure of ME3 in 2022, significant strides have been made in characterizing the structure of this enzyme family, establishing a basis for subsequent small-molecule drug development [36,38].

Fig. 6.

The quaternary structure of MEs and their inhibitors

(A) The various subtypes of human MEs share a similar quaternary structure, forming a homotetrameric protein. The quaternary structure of proteins shown inFig. 6A was obtained from the Protein Data Bank (ME1: PDB ID7X11, ME2: PDB ID1DO8, ME3: PDB ID8E76). (B) Chemical molecular structures of ME inhibitors.

The role of MEs in mediating tumor progression and drug resistance has garnered considerable interest in recent years. Therefore, targeting MEs could potentially become a novel approach in cancer therapy (Fig. 6B–Table 2). A virtual screening against homologous models of MEs conducted in 2006 led to the design and synthesis of novel ME inhibitors based on a piperazine-1-pyrrolidine-2,5-dione scaffold [105]. AS1134900 is a small-molecule inhibitor targeting ME1 (with an IC₅₀ value of 0.73 μM) with high selectivity. Upon binding, it reduces the flexibility of domain C of ME1 and competitively inhibits its activity in the presence of substrates NADP⁺ and malate [106]. However, it fails to effectively inhibit the proliferation of pancreatic cancer cells, possibly due to its limited cell permeability. Optimization of this inhibitor could offer new directions for ME1-related cancer therapy in the future [106].

Table 2.

ME inhibitors.

ME	Inhibitors	Cancer types	IC50(μM)	Target site	Advantages	Disadvantages	Ref
ME1	AS1134900	Pancreatic cancer	0.73	Between the α-helices of domains B and C	High specificity	Low cell permeability; Requiring further optimization	[106]
ME2	NPD389	/	4.63 ± 0.36 (No Brij-35), 5.59 ± 0.38 (0.01 % Brij-35)	/	Fast-binding	Requiring NAD+ for binding; Mixed-type inhibition	[107]
ME2	NPD387	/	11.84 ± 0.57 (No Brij-35), 18.27 ± 1.46 (0.01 % Brij-35)	/	Novel structure	Lower efficiency; Structural instability	[107]
ME2	MDSA	Lung cancer	0.51	The dimer interface's allosteric fumarate-binding site	High specificity	Varying efficacy	[27]
ME2	EA	Lung cancer	0.1	The dimer interface's allosteric fumarate-binding site	High specificity	Limited inhibition	[27]
ME2	Na2EA	Acute myeloid leukemia	120	/	High specificity	Dose-dependent effects	[77]
ME3	Several analogs of 6-piperazin-1-ylpyridin-3-ol amides	Pancreatic cancer	0.06–0.58	Compound 10b binds the hydrophobic pocket of ME3	High specificity; Stable tetramer structure	Lacking allosteric regulation	[108]

ME2 inhibitors have garnered popularity in recent years. In 2014, a 2,5-dihydroxyl benzoquinone derivative, NPD387, was identified through high-throughput screening as a moderate ME2 inhibitor. It exhibits specificity by binding to the 2,5-dihydroxybenzoquinone skeleton of ME2 [107]. However, it has lower efficacy and structural instability. A more potent derivative, NPD389, was also discovered, which binds rapidly and non-competitively to the NAD⁺ binding site of ME2. NPD389 has fast-binding properties, is a highly effective inhibitor, and shows specificity for ME2. Nevertheless, it requires NAD⁺ for binding and demonstrates mixed-type inhibition [107]. Two ME2-specific inhibitors, 5,5′-methylenedisalicylic acid (MDSA) and embonic acid (EA), were reported to inhibit ME2 activity in non-small cell lung cancer cells (with IC₅₀ values of 0.51 μM and 1.1 μM, respectively), leading to tumor cell death [27]. Specifically, MDSA and EA bind to the fumarate binding site of ME2 and inhibit ME2 by inducing conformational changes, disrupting cellular energy metabolism, and inhibiting the migration and invasion abilities of non-small cell lung cancer cells [27]. The fumarate binding site is located at the dimer interface of the enzyme, approximately 30 Å from the active site [40]. Similarly, disodium salt of embonic acid (Na2EA) inhibits ME2 activity by triggering conformational changes, disrupting redox homeostasis, reducing energy metabolism, inducing apoptosis, and inhibiting tumor growth in acute myeloid leukemia, suggesting that targeting ME2 could be an effective strategy for treating acute myeloid leukemia [77]. Moreover, treatment effectively reduces ME2 activity and ATP levels in non-cancerous cells, demonstrating that ME2 inhibitors impact the energy metabolism of both cancerous and normal cells [27]. This emphasizes the necessity for the future advancement of ME2 inhibitors that specifically target tumor cells while minimizing effects on normal cells [27].

ME3 inhibitors have recently emerged. Several analogs of 6-piperazin-1-ylpyridin-3-ol amides possessing potent inhibitory activity against ME3 have been synthesized and identified [108]. Notably, the in silico binding analysis of compound 10b showed that ring B was aligned toward the hydrophobic pocket of ME3. Furthermore, analogs of AS1134900 (ME1 inhibitor) are speculated to be effective against ME3, based on the premise that ME3 has a non-allosteric structure and an AS1134900-binding site that is highly conserved between ME1 and ME3 [38].

In summary, research on ME inhibitors is still in its infancy. In the face of several challenges, further in-depth research on MEs is expected to assist future development activities.

7. Conclusions and perspectives

MEs are metabolic enzymes located in the cytoplasm or mitochondria of cells. They drive various critical physiological metabolic processes in both eukaryotes and prokaryotes. A close association has been identified between MEs in mammalian cells and the onset and progression of cancer. This review has detailed the molecular regulatory mechanisms and biological functions of MEs in cancer and summarized recent research on their involvement in tumor drug resistance and the development of inhibitors. At the molecular level, MEs first influence the cellular redox balance, proliferation, and metastatic ability of tumors by inducing transcriptional changes in the expression of several key molecules. Subsequently, MEs affect post-transcriptional modifications through miRNAs, altering the energy metabolism programming, proliferation, and migration abilities of cancer cells. Post-translational modifications of MEs are complex and interconnected. A dynamic link exists between phosphorylation and acetylation, whereas methylation and desuccinylation alter mitosis or mitochondrial function. MEs can also directly bind to proteins or form complexes, linking multiple metabolic pathways and thus influencing tumor progression. In terms of biological functions, we highlight the role of MEs in tumor energy metabolism reprogramming, programmed cell death, senescence and cell cycle progression, and TME modulation. While MEs predominantly exhibit a pro-carcinogenic effect in most malignant tumors, they also play a role in suppressing the growth of certain tumors.

By regulating cell death pathways, redox balance, and energy metabolism, MEs can also influence the extent to which tumor cells are resistant to chemotherapeutic drugs, such as cytarabine, targeting agents like sorafenib and osimertinib, and the iron-induced cell death inducer ACXT-3102. The multidimensional role of MEs in malignant tumors makes them potential targets for cancer therapy. Consequently, research on ME inhibitors has been on the rise. These inhibitors can competitively inhibit ME1 activity or allosterically inhibit ME2 activity by binding to its malate-binding site. Overall, research on MEs and malignant tumors is still in its early stages, and the application of MEs-based therapy in clinical settings faces several challenges. However, gaining a deeper understanding of its specific molecular mechanisms and biological functions, and emphasizing the role that MEs play in tumorigenesis, will highlight the therapeutic potential of using ME inhibitors in cancer management. Future in-depth studies on the mechanisms of MEs, multidisciplinary integration, and the availability of clinical trial evidence will contribute to optimizing cancer treatment modalities, ultimately improving the efficacy of cancer treatment for patients.

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CRediT authorship contribution statement

Huan Wang: Writing – original draft, Funding acquisition. Wanlin Cui: Writing – original draft. Song Yue: Investigation. Xianglong Zhu: Investigation. Xiaoyan Li: Investigation. Lian He: Investigation. Mingrong Zhang: Investigation. Yan Yang: Investigation. Minjie Wei: Writing – review & editing. Huizhe Wu: Writing – review & editing. Shuo Wang: Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

6PGD

6-phosphogluconate dehydrogenase

ACAT1

acetyl-CoA acetyltransferase 1

ACSS2

acetyl-CoA synthetase short-chain family member 2

AKT1

AKT serine/threonine kinase 1

AMPK

AMP-activated protein kinase

dNTP

deoxy-ribonucleoside triphosphate

EA

embonic acid

ECAR

extracellular acidification rate

ETV4

ETS variant transcription factor 4

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

HMGB1

high-mobility group box 1

HTC

hydride transfer complex

KRAS

kirsten rat sarcoma viral oncogene homolog

LDHA

lactate dehydrogenase A

LKB1

liver kinase B1

MDH1

malate dehydrogenase 1

MDM2

murine double minute 2

MDSA

5,5′-Methylenedisalicylic acid

ME

malic enzyme

ME1

malic enzyme 1

ME2

malic enzyme 2

ME3

malic enzyme 3

MEs

malic enzymes

miRNA

microRNA

mtDNA

mitochondrial DNA

mTORC1

mammalian target of rapamycin complex 1

MYC

MYC proto-oncogene, bHLH transcription factor

Na2EA

disodium salt of embonic acid

NEK1

NIMA-related kinase 1

NRF2

nuclear factor erythroid 2-related factor 2

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

OCR

oxygen consumption rate

PC

pyruvate carboxylase

PFKL

phosphofructokinase

PGAM5

PGAM family member 5

PKM2

pyruvate kinase M2

PRMT1

protein arginine methyltransferase 1

ROS

reactive oxygen species

SIRT5

sirtuin 5

SLC7A11

solute carrier family 7 member 11

SREBP-1

sterol regulatory element binding protein 1

TCA

tricarboxylic acid cycle

TME

tumor microenvironment

Data availability

No data was used for the research described in the article.