Introduction
In February 2022, the China National Cancer Center indicated that the incidence of oral cancer in 2016 was 3.78 cases per 100,000 individuals annually [
1]. Oral adenoid cystic carcinoma (OACC), accounting for less than 2% of malignant head and neck tumors (3–4.5 cases per million annually worldwide), is a rare tumor of oral cancer [
2]. Due to its rarity, limited research has been conducted on the pathogenesis and treatment of OACC. OACC is distinguished by a significant occurrence of local–regional recurrence and distant metastasis. The survival rate of OACC within a span of 15 to 20 years is approximately 23 to 40% [
3,
4]. Recurrence rates within the 5-to-10-year range vary from 30 to 75% [
5]. To date, drug treatment for OACC have not been standardized and the progress on targeted treatment has been sluggish. The standard treatment options for OACC in clinical practice involve surgical intervention and radiotherapy [
6,
7]. However, these interventions have a significant negative impact on patient’s quality of life, including changes in saliva volume, chewing ability, facial appearance, and verbal expression. Consequently, it is crucial to explore the pathogenesis of OACC and identify new targeted treatments.
Human genes constitute approximately 30,000 entities, of which approximatively 2% encode proteins. But protein post-translational modifications (PTMs), involving specific chemical alterations, have resulted in the formation of approximately two million protein entities [
8]. Previous studies have identified more than 400 different types of PTMs that play crucial roles in numerous cellular functions, including cellular proliferation, metabolism, and signal transduction [
9]. PTMs are taking part in various biological processes and closely associated with the pathogenesis of various tumors [
10].
Khib was initially identified as a novel post-translational modification (PTM) on histones in HeLa and mouse testis cells by Dai et al. [
11]. It is characterized by the addition of a 2-hydroxyisobutyryl group from the donor molecule 2-hydroxyisobutyryl-CoA to its target protein. Subsequent studies have showed the wide distribution of Khib in both prokaryotes and eukaryotes. With scientist’s efforts, the relationship between Khib and several cancer types has been unveiled gradually. Zhang et al. discovered significant alterations in Khib modification levels within the actin cytoskeleton regulatory pathway in oral squamous cell carcinoma (OSCC), highlighting the importance of Khib in OSCC pathogenesis [
12]. Furthermore, Yuan et al. found that cell proliferation in liver malignant tumors could be suppressed by inhibiting the Khib modification levels of ENO1K281 [
13]. These findings have motivated this study into the potential association between Khib and OACC, which may pave a new way for understanding OACC mechanisms and developing new targeted treatment.
Through a comprehensive investigation of the proteome and post-translational alterations in OACC, we unraveled the potential role of Khib in driving the progression of this malignancy. This study elucidated the influence on the enzyme activity within the glycolysis pathway, shedding light on a plausible mechanistic explanation for OACC advancement.
Discussion
Numerous previous studies have demonstrated that Khib participated in diverse biological activities, including glycolysis. Wu et al. conducted a comprehensive analysis of Khib proteome sites in lung cancer cell globally, revealing alterations in ten pivotal glycolysis enzymes due to Khib modification. Among these enzymes, seven experienced substantial modifications with over ten Khib residues [
16]. Additionally, Huang et al. determined that the reduction of Khib levels on pivotal glycolysis enzymes significantly impairs their activities. Compared to the control group, hypo Khib-modified cells display notably lower intermediate metabolite concentrations in the glycolysis pathway, indicating decreased enzyme function [
17].
Cancer cells’ abnormal energy metabolism may affect several associated metabolic pathways, influencing many biological processes for satisfying their increased growth demands and survival under a range of stress circumstances [
18]. Regulation of the glycolysis pathway is one of the “hallmarks of cancer.” Consequently, targeting glycolysis remains attractive for therapeutic intervention. For example, Xu et al. revealed that Chrysin suppressed the glycolysis pathway by reducing HK-2 in tumor tissue, disrupting the energy supply required for tumor growth and inhibiting tumor cell proliferation [
19].
With cutoff conditions of fold change (FC) ≥ 1.2 (log2-fold change ≥ 0.26) or ≤ 1/1.2 (log2 fold-change ≤ − 0.26), a total of 2011 Khib sites were identified on 764 proteins. We conducted a systematic bioinformatics analysis for illustrating Khib landscape in OACC. The glycolysis/gluconeogenesis pathway was the most significantly enriched KEGG pathways of both hyper-DHMPs and hypo-DHMPs in this study. In the glycolysis pathway, all 10 pivotal enzymes were modified by Khib (Fig.
10, Supplementary Table
10). Several studies have shown that acetylation is crucial for stability in the glycolytic pathway, while the mechanisms of other PTMs, such as Khib, have rarely been reported.
Hoper-DHMPs and hypo-DHMPs were analyzed by Cytoscape plugin cytoHubba based on stress. This study has identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase 1 (ENO1) as prominent constituents among the top 10 proteins (Supplementary Table
7). GAPDH, exchanging glyceraldehyde-3-phosphate (G3P) for 1,3-biphosphoglycerate (1,3-BPG) [
20], is essential in aerobic glycolysis of numerous cancers [
21‐
23]. In this study, Khib of GAPDH was hyper-modified at K254 (fold change: 1.29). Chen et al. conducted an analysis of GAPDH acetylation levels and measured GAPDH enzyme activity proposing that hyper-modified acetylation of GAPDH at K254-enhanced GAPDH enzyme activity and tumor cell proliferation [
24]. Khib of GAPDH in K254 may play a critical role in the glycolysis pathway to promote cell growth and tumorigenesis. ENO1, a high-energy intermediate, facilitates the conversion of 2-phosphoglycerate to the phosphoenolpyruvate (PEP). ENO1 is intricately linked to the pathogenesis of cancer. Previous study showed that the FAK/PI3K/AKT pathway whose downstream signals could elevate the glycolysis to significantly facilitate non-small cell lung cancer proliferation and metastasis would be activated by upregulated ENO1 [
25]. In this study, the Khib in ENO1 showed upregulation trends at 15 sites and K228 was one of the most significant hyper-modified sites (fold change: 3.59). K228 is the key residues that mediate ENO1 catalytic activity [
26]. The recent study revealed that lysine residue K228 was on the ENO1 surface, rendering them susceptible to modification by Khib [
17]. When a bulky and hydrophilic 2-hydroxyisobutyryl group replaces the positively charged lysine side chain at K228, it may disrupt cofactor binding and induce conformational changes that impact substrate binding and turnover. Consequently, the activity of ENO1 is enhanced in response to the heightened glucose consumption associated with cancer malignancy [
13,
17]. Based on this premise, we hypothesized that K228hib also elevated glycolysis in OACC cells by augmenting the activity of ENO. The previous study showed that acetylation of ENO at lysine residue 257 can neutralize its positive charge and change binding site geometry, thereby disturbing the electrostatic binding potential and impairing the enzyme’s ability to bind substrates, ultimately inhibiting the glycolysis pathway [
27,
28]. In this study, a noteworthy observation was made regarding the sole and significant downregulation of K256hib in ENO1. This residue is located in close proximity to K257 and may possess a similar functionality to K257. K256hib potentially exerted an inhibitory effect on the binding capacity of ENO1 by inducing alterations in the shape of the binding site and the electrostatic binding potential. We postulated that upregulation of K228hib and downregulation of K256hib both increased the catalytic efficiency of ENO1 to promote glycolysis pathway which favor OACC progression.
Phosphoglycerate kinase 1 (PGK1) is one of the top ten hub proteins of hoper-DHMPs based on Cytoscape cyto-Hubba analysis (Supplementary Table
7). PGK1 which catalyzes the reversible transfer of a phosphoryl group from 1, 3-bisphosphoglycerate (1, 3-BPG) to ADP and produces 3-phosphoglycerate (3-PG) and ATP [
29] is composed of N- and C-terminal [
30]. The N-terminal domain of PGK1 allowed 1,3-BPG or 3-PG to bind, and the C-domain allowed ADP to bind [
31]. We found that all Khib sites of PGK1 are upregulation trends in OACC-tumor tissues, including the K323 site (fold change: 1.33). According to previous studies, acetylation at K323, located in its C-terminal domain of PGK1, enhanced its catalytic efficiency by increasing its affinity for ADP/ATP [
32] and promoted the proliferation, glycolysis, and tumorigenesis of liver cancer [
33]. Thus, we supposed that hyper Khib-modified of PGK1 at the K323 site may also enhance proliferation and tumorigenesis of OACC by enhancing the binding affinity of ADP/ATP to promote PGK1 activity and glycolysis.
Hexokinase (HK), the first rate-limiting enzyme in the glycolysis pathway, plays a critical role in cancers. In instances where tumor cells experience a severe glucose deficit, HK1 assumes a more prominent role in facilitating glycolytic reactions compared to HK2, owing to its lower Km. The expression of HK1 has been shown upregulated trend which is beneficial to cancer cell proliferation by enhancing glycolysis in some cancer types, including pancreatic malignant tumor [
34], bladder carcinoma [
35], and ovarian malignant tumor [
36]. In this study, compared with OACC-N, the expression of HK1 is upregulated in OACC-T. This upregulation serves to promote glycolysis, thereby enhancing oral proliferation and migration of OACC. It is worth noting that all Khib of HK1 are upregulation trends in OACC-tumor tissues, especially at K488 (fold change:5.39) and K187 (fold change: 2.43). Zhang et al. reported that phosphorylation of Y732 probably improved the catalytic efficiency of HK1 by causing the homodimerization of the enzyme to break down, which raised the enzyme’s affinity for the substrate glucose [
37]. To date, a limited number of studies contain data on the relationship between Khib of HK1 and its enzyme activity, necessitating further investigation in this domain. We supposed that hyper Khib-modified of HK1 probably also caused the homodimers to separate, thereby increasing catalytic efficiency of HK1 and enhancing glycolysis, ultimately facilitating tumorigenesis and metastasis. Pyruvate dehydrogenase A1 (PDHA1) belongs to the enzyme complex known as pyruvate dehydrogenase complex, which acts a gate-keeper enzyme between the mitochondrial citric acid cycle and glycolysis. Pyruvate dehydrogenase is crucial for the cancer metabolism and its suppression in cancer cells can boost the Warburg effect. Previous studies have shown a significant downregulation of PDHA1 expression in breast cancer [
38], ovarian carcinoma [
39], and gastric cancer [
40] resulting in enhanced glycolysis and an association with poor prognosis. However, the correlation between the PDHA1 protein expression and the metabolism and biological behavior of OACC is unclear. In this study, PDHA1 was underwent significant down trend expression (fold change: 0.76), which suggest that the metabolic change in OACC cells to rely more on glycolysis may be due to attenuated mitochondrial function through inhibition of PDHA1. In conclusion, targeting Khib may be an attractive OACC therapy.
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