Protein Advanced Glycation End Products and Their Implications in Pancreatic Cancer

Protein glycation is a posttranslation modification (PTM) that is etiologically relevant to the pathogenesis of various diseases, including cancer (1–3). The aldehyde or ketone groups of reducing sugars, such as glucose and fructose, can spontaneously react with lysine and arginine residues in proteins through the Maillard reaction to form advanced glycation end products (AGE) with various structures.

In the conditions of diabetes, high exposure of exogenous sugar or AGE sources (e.g., highly processed meat), aging or malignancy, formation and accumulation of protein AGEs in the human body can be a proteome-wide phenomenon. Due to their potential noxious effects, AGEs are also called glycotoxins (4). Protein glycation and abnormal accumulation of protein AGEs can have a direct and profound impact on the intracellular proteome, including the dysregulation of protein functions and expression, protease resistance, protein aggregation and turnover, and stress on cellular machinery for clearance of impaired proteins (refs. 5–10; Fig. 1A). In addition, AGEs can activate the receptor for AGEs (RAGE; ref. 11), which mediates pro-inflammation and oxidative stress, leading to the stimulation of cancer associated-pathways, including NF-κB, AP-1, and STAT (12–14), and DNA damage (refs. 15, 16; Fig. 1B). Altogether, the broad negative impacts of protein AGEs, RAGE, and their significant link with premature manifestation of cancer and other complex diseases in the modern environment have been increasingly recognized in recent years (17, 18). Currently, while significant interests have been focused on investigating the biology underlying AGE–RAGE stimulation and RAGE-dependent pro-inflammatory signaling cascades, the intrinsic implication of intracellularly formed protein AGEs in regulating cellular functions and signaling has been an underappreciated process in AGEs-related diseases. These proteome impairments can induce a cascade of alterations in cellular proteome and functions, leading to homeostatic imbalance, and eventually causing health problems or disease development, including carcinogenic transformation towards development of neoplasms.

Pancreatic ductal adenocarcinoma (PDAC) accounts for 80% to 90% of pancreatic cancer cases. It is a lethal disease with a 5-year survival rate of 12% in the United States, the lowest among solid cancers (19). Diabetes mellitus, aging, cigarette smoking, obesity, high dietary intakes of sugar and highly processed meat are known risk factors for PDAC development. It is not a coincidence that most of these PDAC risk factors are the major endogenous or exogenous sources for glycative stress and the formation of protein AGE adducts in the human body. Collective clinical and epidemiologic evidences have suggested the link between the accumulation of protein AGEs and neoplastic changes (3, 6, 7, 20, 21). The implications of protein AGEs in pancreatic carcinogenesis are multifactorial and throughout PDAC progression. Despite its role in promoting PDAC development being increasingly recognized clinically and epidemiologically, knowledge gaps exist in understanding the molecular details that govern the formation of protein AGE adducts and their implications in proteome impairment and downstream signaling cascades linked to PDAC development. These knowledge gaps have hampered the translational efforts to develop clinical applications for risk assessment, prevention and intervention of PDAC for the large at-risk populations.

Diabetes mellitus has a major contribution to protein glycation, especially in blood proteins. In diabetic or hyperglycemic conditions, insulin resistance can impair glucose metabolism and result in the higher level of glucose in the blood, which can then react with proteins and form protein AGEs. Clinical and animal studies conducted on patients with diabetes (22, 23) and mice with diabetes (24) have shown increased serum AGEs levels.

Mounting efforts have been made to shed light on the molecular events linking protein AGEs with pancreatic carcinogenesis and progression, such as proteome impairment and dysfunctions (8, 25), as well as the involvement of AGE–RAGE axis and associated signaling cascades in inflammatory and metabolic pathways (26–29). Collectively, these studies have suggested the functional roles of protein AGEs as a multifactorial mediator in the complex interplays between diabetes and PDAC. A recent epidemiologic study, which investigated the association of diabetes with the incidence of 16 cancers among 378,253 individuals, found a positive association between hyperglycemia (based on HbA1c test) and PDAC (30). Having diabetes for a long period of time could be a predisposing factor for the development of sporadic PDAC by promoting tumorigenesis through the cancer stemness and epithelial-mesenchymal transition (31–33). Abnormal accumulation of protein AGEs due to long-term hyperglycemic condition may induce proteome impairment, reactive oxygen species (ROS) and AGE–RAGE driven pro-inflammatory signaling events to promote cancer hallmarks.

Notably, studies have also shown that individuals with new-onset diabetes within 3 years are having a greater prevalence to be diagnosed with PDAC (34, 35). An existing PDAC tumor, which has high levels of glycolysis in cancer cells, can result in the formation and accumulation of protein AGEs in the pancreas to cause pancreatic β-cell injury and impair insulin secretion (36–38), and consequently induce hyperglycemia or diabetes. At its asymptomatic stages, PDAC induced secondary (pancreatic) diabetes may be related to new-onset adult diabetes which is later diagnosed with PDAC. As the disease progresses, invasive PDAC can obstruct pancreatic ducts, causing pancreatitis and exacerbating diabetic conditions (39). The implications of protein AGEs in the pathogenesis of both diabetes and PDAC may afford crucial information to elucidate the complex interplays between the two diseases, and offer a unique opportunity for prevention and intervention of PDAC among diabetics.

Dietary intake of exogenous AGEs and simple sugars, as well as cigarette smoking are important contributors to the glycative stress and formation of protein AGEs in the human body (40, 41). Dietary-derived AGEs exogenously generated from highly processed food, such as red meat cooked with high temperature, can be absorbed by the intestine, accumulated within cells and tissues, and presented in the circulating system (40, 41). Although only a proportion of the exogenous AGEs and their precursors was remained in the human body, a study had found that AGEs (e.g., hydroimidazolone MG-H1 residues) absorbed from food were substantially enriched in blood plasma (42). Studies have shown that pancreatic β-cells are highly susceptible to the oxidative stress promoted by dietary AGEs, which can cause defects in insulin secretion as well as β-cells death (43). With excess consumption of dietary AGEs, these exogenously introduced free AGE adducts can accumulate and stimulate the AGE–RAGE axis and induce systemic oxidative stress and chronic inflammation, leading to various diseases (20), including PDAC (26, 44).

Apart from the protein AGEs formed endogenously due to diseases, dietary sugar is another major source for protein AGE formation and accumulation in vivo. It has been reported that sugar-sweetened food and beverages can introduce markedly high levels of dietary-derived protein AGEs (40, 41). Due to the consumption of highly sugary diets, the reducing sugars, such as glucose and fructose, are taken up by cells and react with proteins to form intracellular protein AGEs. The endogenous formation of protein AGEs occurs from days, weeks to months and consequently can affect long-lived proteins. The metabolisms of the monosaccharides undergo different pathways, and can have various impacts on protein AGE formation. In general, glucose and sucrose have higher absorptive capacity than fructose, as glucose can be metabolized to produce energy in various organs while fructose is primarily metabolized in the liver (45). Conversely, in terms of kinetic in generating glycation precursors, fructose, which has a highly reactive open chain form, is much more reactive and has a substantially higher glycoxidation rate than that by glucose, which has a relatively stable ring structure (46, 47). Studies have suggested that among the commonly used dietary sugars, fructose might represent the most hazardous for protein AGE formation and accumulation (40, 48). The metabolic pathways of glucose and fructose in the cells are illustrated in Fig. 2. The rate limiting step in the glucose pathway is the convention of glucose-6-phosphate to fructose-1–6-diphosphate regulated by the phosphofructokinase. On the other hand, fructose is phosphorylated by ATP to fructose-1-phosphate catalyzed by fructokinase. Notably, glyceraldehyde is the common metabolic intermediate product of both glucose and fructose, and is highly reactive to form protein AGEs and can cause toxic effects directly on cells (9). Because glucose, fructose, and other reducing sugars are carried into the human body through dietary consumption, some amount of glycative stress is present even in healthy individuals. However, unhealthy dietary habits can result in abnormal accumulation of protein AGEs and high glycative stress in the human body, leading to glycation of insulin & proinsulin, mitochondrial dysfunction, ROS induced β-cell dysfunction & death, upregulation of inflammatory pathways and other negative impacts on pancreas linking to PDAC (20).

Cigarette smoke contains many carcinogens, including reactive glycation species that can rapidly react with proteins to form endogenous AGEs. Because tobacco has a considerable amount of reactants and curing of tobacco products leads to Maillard reaction, smoking significantly contributes to the protein AGEs formation. Reactive carbonyls are considered the major reactants of cigarette smoke to form protein AGEs (49, 50). In addition, oxidative stress and changes in metabolic processes resulting from aging are linked to the abnormal accumulation of AGEs and their functionally compromised protein adducts (51). Such AGEs-related protein impairments can have a functional damage on multisystem and contribute to the aging phenotype and the development of many aging-related diseases (52), including PDAC. Much remains to be learned about the biology of causal nature of protein AGEs attributed to environmental factors and aging to PDAC risk, and whether new interventions might be developed to prevent or reduce the negative impact of protein AGEs-related cellular damage.

The formation and accumulation of protein AGEs negatively impact the structure and functions of extracellular and intracellular proteins, which consequently lead to cellular malfunction and disruption of cellular homeostasis. Glycation also affects immunity because many immunoglobulins can be modified with the glycation reaction, which reduces their functional capability. Studies have shown that glycation of immunoglobulin G impairs the Fc fragment function and the activity of complement activation (5, 53). Histones are proteins with rich lysine and arginine composition and are very prone to be glycated. Many studies have shown that histones can be glycated and cause epigenetic changes to negatively influence gene transcription (54–56). Glycation occurring on enzymes can alter the conformation of the enzyme's active site, which leads to the impairment of substrate binding. The antioxidant properties of certain enzymes, such as superoxide dismutase and glutathione reductase, can also be affected due to protein glycation and AGE formation (57). Pancreas is the organ that produces the majority of the digestive enzymes. Abnormal formation of protein AGEs on these enzymes through hyperglycemia or dietary sugars may impact the physiologic activities of the pancreas, thus induce genomic and proteomic dysregulations on these enzymes as well as their associated protein functional networks.

The addition of sugar molecules to the proteins through glycation has been reported to cause structural modification and dysfunction of the proteins, which ultimately facilitate protein aggregation (58, 59). Recent research findings revealed that protein aggregation also contributes to the development of cancer (58). The glycation mediated aggregation and functional inactivity of tumor-suppressor proteins, such as p53, may potentially promote pancreatic cancer risk and development (60, 61). Studies have shown that glycation of proteins results in the formation of globule-like deposits (59, 62). Due to the formation of covalent bonds between AGEs, oxidation of sulfur groups to form disulfide bridges, and formation of new reactive groups in the proteins, protein aggregation is facilitated through glycation process (5). In addition, it has been suggested that glycation of positively charged lysine and arginine residues changed their net charge, resulting in the exposure of hydrophobic residues to the surface (63). These hydrophobic surfaces tend to clump and form protein aggregates, which leads to the ER stress, and ultimately triggers the unfolded protein response to activate ubiquitin-mediated proteasome system (UPS) and autophagy, the two major intracellular degradation pathways (64). These AGEs-modified and -aggregated proteins are resistant to cleavage by proteolytic enzymes (65) and can escape from the clearance by UPS (66). Thus, autophagy plays an important role in clearing the accumulated protein AGEs (64, 67). In addition, glyoxalase system also serves as a detoxification machinery by converting highly reactive methylglyoxal formed during sugar metabolism into far less reactive D-lactate, thus preventing protein AGEs formation (68, 69). Protein aggregation and protease resistance resulted from abnormal accumulation of protein AGEs can therefore further induce imbalance in homeostasis of cellular clearance mechanisms, such as autophagy, which has multifactorial functional roles in cancer development and progression (70) and is profoundly implicated in PDAC (71).

It is known that protein AGEs accumulation induces ROS generation, which can create a dose dependent multifactorial effects on cells, including loss of molecular integrity, disruption in cellular signaling and homeostasis, followed by inflammation and tissue injury. It has been reported that the accumulation of extracellular AGEs resulted in pancreatic β-cells impairment and reduction in insulin secretion mainly through the oxidative stress mediated by mitochondrial pathway (38). In tumorigenesis, ROS plays a dual role, where it promotes cell survival and proliferation at low or moderate level, and on the other side it can cause cell death at high level (72). In the physiologic conditions, because the hyperglycemia- or dietary-introduced protein AGEs formation and accumulation are not an acute process, the AGE-induced ROS generation may also occur slowly to retain a moderate level of ROS. As such, the moderate level of ROS induced by protein AGEs may favor tumorigenesis and progression. It has been reported that ROS induced by high glucose stimulated the proliferation of pancreatic cancer cells by suppressing the JNK pathway (73). Furthermore, intracellular accumulation of protein AGEs can have an environmental influence on the surrounding cells to stimulate cancer signaling, such as oxidative stress and AGE–RAGE driven pro-inflammatory events. Such an impact may be through the release of intracellularly glycated amino acids or free AGE adducts into the neighboring environment via multiple routes, including apoptosis, protein turnover by cellular proteolysis, as well as secretion of extracellular vesicles.

The RAGE is a transmembrane protein and a multiligand receptor. In addition to AGEs, several other ligands, such as some proteins of the S100 family, β-amyloid peptides and β-sheet fibrils, high-mobility group box 1 (HMGB1), and prions can bind with RAGE to mediate the inflammatory cellular signaling towards cancer development (12, 74). By binding AGEs to the RAGE, several signaling pathways are activated, including Ras-extracellular signal-regulating kinase-1/2, p38 mitogen-activating kinase, ERK-1/2, JNK, NADPH-oxidase, and JAK1/2 (12–14). These signaling pathways ultimately activate transcription factors STAT family, AP-1 and NF-κB to induce target gene expression (12–14). RAGE expression is relatively higher in tumor cell lines derived from pancreas compared with other tumors, such as melanoma, fibrosarcoma, colonic, and breast carcinoma (75).

It has been demonstrated that AGE–RAGE activation promotes PDAC progression via promoting the pro-inflammatory signaling, which favors the tumorigenesis (24–28, 75–77), as oxidative stress, chronic inflammation and cancer are fundamentally linked. The expression of RAGE in PDAC cells limited apoptosis and upregulated autophagy to promote cancer cell survival (75). It has been reported that RAGE expression is higher in mesenchymal tissues than parenchymal tissues in rat pancreas; and rat pancreatic fibroblasts have a major contribution to AGE–RAGE signaling and inflammation compared with the parenchymal beta cells (78). The AGE–RAGE interaction induced the phosphorylation of JNK, p38, AKT, IKKα/β, and NF-κB in rat pancreatic fibroblasts, and consequently, upregulated the mRNA expressions of cytokines, such as IL1β and IL6 (78).

Nε-carboxymethyl-lysine treatment in pancreatic cancer cells has been shown to activate the NF-κB signaling and subsequently increased the cell proliferation (76). The same study found that AGE-stimulated RAGE expression promoted PDAC cell growth in a concentration- and time-dependent manner and was implicated in PDAC development. Exogenous administration of AGEs to PDAC-prone mice induced RAGE upregulation in pancreatic intraepithelial neoplasia (PanIN) and markedly accelerated progression to invasive cancer (76). Genetic deletion of RAGE in mouse models of PDAC progression showed a significant delay in the development of PanINs and a prolongation in PDAC survival (79). The use of RAGE antagonist peptide to counteract RAGE activation also delayed PanIN development in CML-treated PDAC mouse model (76). Soluble RAGE (sRAGE) competitively bound with AGEs and prevented the AGE–RAGE interaction and pro-inflammatory signaling cascade, and was inversely association with pancreatic cancer risk (80–82). Overexpression of cellular RAGE has been shown to promote the pancreatic cancer progression (83). Table 1 summarizes some studies in revealing the implication of protein AGEs and RAGE in pancreatic carcinogenesis.

Protein AGE adducts include a cohort of heterogeneous modifications involving amino groups in lysine residues and N-terminal or guanidino groups in arginine residues. Table 2 exemplifies some common non–cross-linking AGEs with well-characterized structures.

Traditional methods, such as spectrophotometry and immunoassays, have been widely used to measure the “total AGEs”. Spectrophotometric methods using absorbance or fluorescence do not provide information about individual AGE classes. While immunoassays can target certain AGE forms, antibody-based techniques suffer from a high degree of nonspecific binding and typically do not allow multiplexed analysis of protein AGEs. Earlier gas chromatography–mass spectrometry or liquid chromatography-mass spectrometry approaches, relying on exhaustive protein hydrolysis with subsequent amino acid analysis, also suffer from various technical difficulties, and do not provide protein glycation site-specific AGE structural information.

Mass spectrometry (MS)-based proteomics is currently the best available tool to meet the challenges for proteome-wide protein AGE adduct analysis. In shotgun proteomics, one of the main challenges for glycated peptide analysis is the weak fragmentation of the peptide backbone. High abundance ions corresponding to various neutral-losses of water and sugar moiety can make it difficult to obtain b- and y- ion information for reliable identification of the glycated peptide sequence (84, 85). Soft fragmentation techniques, such as electron-transfer dissociation, can improve peptide fragmentation and facilitate the elucidation of glycated peptide sequence, but suffer low fragmentation efficiency (85, 86). Using high-energy collisional dissociation, studies have demonstrated improved fragmentation for identification of early glycation products (87) and protein AGEs (8). To enhance analytical sensitivity, boronate affinity chromatography has been used to enrich glycated peptides from various biological samples, as cis-diol groups of glycated peptides interact with boronic acids (85).

In addition to the identification of the AGE structures conjugated at specific glycation sites in proteins, quantification of glycation level can provide additional insightful information on protein AGE adducts. Label-free approach has been the most widely applied method in global proteomic analysis of protein glycation or protein AGEs. Isotopic (e.g., SILAC) or isobaric labeling (e.g., TMT, iTRAQ) of amino acids in proteins can also be applied to facilitate the quantitative analysis of protein glycation level. In addition, isotopic labeling of reducing sugar molecules, which enables the distinction of the induced protein AGEs from native glycated species, has been used to study the formation of protein glycation and AGEs (8, 88, 89). Using ¹³C-labeled glyceraldehyde to investigate protein AGEs formed in pancreatic normal and cancerous cells, a study found that lysine was more liable to AGE modification compared with arginine, likely due to the nature of its chemical structure (8). This finding might have an implication in developing novel strategies in reducing the formation and accumulation of protein AGEs targeting lysine-conjugated AGEs.

Comprehensive characterization of protein AGEs in a complex biological sample can be a daunting task. More in-depth and breadth coverages of MS analysis in protein glycation and AGEs can be found in many excellent reviews in the literature (85, 90). While challenges remain, especially in the analysis of cross-linked protein AGEs, MS-based proteomics with high resolution and mass accuracy has emerged as a versatile tool for global analysis of glycated proteome.

Mounting efforts have been directed to better understand the pathophysiologic consequences of protein glycation and AGEs on cancer development and progression, and to explore preventive strategy and therapeutic intervention. Many common PDAC risk factors are the major sources of glycative stress and the formation of protein AGEs in the human body. Apart from AGE–RAGE signaling cascades, abnormal accumulation of intracellular protein AGEs can negatively impact cellular proteome and induce intricate mechanisms leading to altered physiologic homeostasis by inducing oxidative stress, inflammation, protease resistance, protein aggregation, unfolded protein response, metabolic responses, aberrant PTM status, gene dysregulation and other molecular events that are susceptible for the carcinogenic transformation towards development of neoplasms. Recent reports have indicated the profound proteome impairment and functional alterations driven by the formation of protein AGEs, and supported the notion that proteome impairment and RAGE stimulation induced by glycation might be directly linked to the etiologies of sporadic PDAC. Various therapeutic interventions are being explored to prevent the endogenous formation and accumulation of protein AGEs (91, 92). Future studies with in-depth and comprehensive data are expected to extend these efforts toward clinical applications. Currently, while significant interests have been focused on investigating the biology underlying AGE–RAGE stimulation and RAGE-dependent pro-inflammatory signaling cascades, the intrinsic implication of intracellularly formed protein AGEs in impairing cellular functions and signaling has been an underappreciated process in AGEs-related diseases, including PDAC. As recent studies have suggested, strategies to reduce the formation and accumulation of protein AGEs, instead of RAGE inhibition, might be suitable for the risk management and prevention of PDAC. Efforts to narrow the knowledge gap in understanding the molecular details in bridging the impaired proteome, dysregulated functional networks and cancer hallmarks initiated upon the formation and accumulation of protein AGEs may provide new insights to inform risk assessment, prevention and early intervention of pancreatic cancer.

R. Chen reports grants from NIH; and grants from the Cancer Prevention & Research Institute of Texas during the conduct of the study. No disclosures were reported by the other authors.

This work was supported in part by the National Institutes of Health grants R01CA180949 (to R. Chen and S. Pan) and R01CA211892 (to R. Chen), the Cancer Prevention & Research Institute of Texas (CPRIT) grant RP210111 (to S. Pan), and the Rochelle and Max Levit endowment fund (to S. Pan).