Pathological implication of protein post-translational modifications in cancer

Abstract

Protein post-translational modifications (PTMs) profoundly influence protein functions and play crucial roles in essentially all cell biological processes. The diverse realm of PTMs and their crosstalk is linked to many critical signaling events involved in neoplastic transformation, carcinogenesis and metastasis. The pathological roles of various PTMs are implicated in all aspects of cancer hallmark functions, cancer metabolism and regulation of tumor microenvironment. Study of PTMs has become an important area in cancer research to understand cancer biology and discover novel biomarkers and therapeutic targets. With a limited scope, this review attempts to discuss some PTMs of high frequency with recognized importance in cancer biology, including phosphorylation, acetylation, glycosylation, palmitoylation and ubiquitination, as well as their implications in clinical applications. These protein modifications are among the most abundant PTMs and profoundly implicated in carcinogenesis.

1. Introduction

Protein post-translational modifications (PTMs) are chemical changes on amino acid side chains or protein terminuses. An enormous number of proteoforms can be generated by more than 200 known PTMs (Deribe et al., 2010; Duan and Walther, 2015; Olsen and Mann, 2013), profoundly influencing the complexity of proteomes and vast biological functions. A proteome-wide PTM study on the Swiss-Prot protein database suggested that the top five most prevalent PTMs observed experimentally were phosphorylation, acetylation, glycosylation, amidation and hydroxylation, whereas the top five putative PTMs were glycosylation, phosphorylation, acetylation, methylation and palmitoylation (Khoury et al., 2011). PTMs constitute a central mechanism that is beyond the genetic code to regulate cellular functional machinery in response to developmental or/and environmental stimuli. The majority of these modifications are catalyzed by various enzymes through highly regulated complex pathways, while some of them are driven by non-enzymatic chemical reactions, reflecting the convoluted influences of both genomic, transcriptomic and environmental factors on PTM status (Harmel and Fiedler, 2018; Trougakos et al., 2013; Walsh et al., 2005).

It is well known that PTMs are profoundly implicated in tumorigenesis and immune modulation, and have been emerging as important targets for early detection and therepeutic treatment of cancer (Benton et al., 2017; Chandler et al., 2019; Chang et al., 2016; Chang and Ding, 2018; De et al., 2014; Gong et al., 2016; Hsu et al., 2015; Jeusset and McManus, 2019; Ko and Dixon, 2018; Lan and Wang, 2019; Leeming et al., 2011; Mereiter et al., 2019; Munkley and Elliott, 2016; Naro and Sette, 2013; Popovic et al., 2014; Resh, 2017; Singh et al., 2017; Watanabe and Osada, 2012; Wu et al., 2019). Figure 1 examplifies various types of PTMs, their influences on protein functions and implications in tumorigenesis. Enzymatic PTMs are pathway driven, and occur at specific motifs in protein sequence, involving various modifications, such as chemical groups (e.g. phosphorylation, acetylation, etc), glycans (glycosylation), lipids (lipidation) or polypeptides (ubiquitination, SUMOylation). While non-enzymatic PTMs (e.g. glycation, oxidation, etc) are conventionally considered as a proteome-wide phenomena, mounting evidences have suggested that their occurrences can also be selective, and influenced by many factors, such as protein structure, amino acid neighboring preference and other local conditions (Harmel and Fiedler, 2018; Trougakos et al., 2013). These modifications can have significant impacts on proteins in many aspects, including activity, stability, functions, structure, localization, trafficking, signaling transduction, and their interactions with partner biomolecules (Chen et al., 2020; Krueger and Srivastava, 2006; Lothrop et al., 2013; Olsen and Mann, 2013; von Stechow and Olsen, 2017). The pathological implications of PTMs are involved in all cancer hallmarks, including sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis (Hanahan and Weinberg, 2000, 2011; Sanchez-Vega et al., 2018) (Fouad and Aanei, 2017; Pan et al., 2020; Senga and Grose, 2021; Wu et al., 2019). PTMs also play essential roles in regulating tumor microenvironment, such as remodeling of extracellular matrix (ECM) for cancer invasion and chronic inflammation (Chandler et al., 2019; Chang and Ding, 2018; Leeming et al., 2011). Targeting PTMs of critical proteins or pathways represents an emerging strategy to improve early detection and therapeutic treatment of cancer.

Fig. 1. Protein PTMs, biological functions and implication in tumorigenesis.

A) Well-known examples of PTMs, B) Influences of PTMs on protein biological functions, C) Pathogenesis implications of PTMs in cancer.

Enormous amount of information has been presented in the literature in studying PTMs relevant to cancer (Brooks and Gu, 2003; Chen et al., 2020; Krueger and Srivastava, 2006; Leeming et al., 2011; Meng et al., 2021; Wang et al., 2020; Wu et al., 2019). With the limited scope and space of this review, we attempt to discuss herein some PTMs of high frequency with recognized importance in cancer biology, including phosphorylation, acetylation, glycosylation, palmitoylation, and ubiquitination, as well as their implications in clinical applications. These protein modifications are among the most abundant PTMs and profoundly implicated in carcinogenesis.

2. Phosphorylation

Phosphorylation is a prevalent and reversible modification on amino acid residues of serine (Ser), threonine (Thr) and tyrosine (Tyr), which are catalyzed by kinases. Many kinases have been identified to be the oncogenic drivers, and their constitutive activities can result in aberrant phosphorylation of target substrates that activate or deactivate signaling pathways implicated in various types of cancer (Brooksbank, 2001; Buday and Vas, 2020; Choudhary and Mann, 2010; Ruprecht and Lemeer, 2014; Ubersax and Ferrell, 2007; Xu et al., 2019). Here we will discuss several driver phosphorylation abnormalities commonly involved in the cancer hallmarks (Hanahan and Weinberg, 2000, 2011).

2.1. RTK/RAS pathway.

The mitogen-activated protein kinase (MAPK) cascades are the major signaling pathways that regulate cellular growth and survival. The main canonical pathway, RTK/RAS/RAF/MEK/ERK pathway, transmits signals through sequential phosphorylation/activation of downstream protein kinases that eventually leads to transcription of genes that encode proteins involved in the regulation of essential cellular functions, including cell proliferation, differentiation and stress responses (Cseh et al., 2014; Santarpia et al., 2012). This pathway is highly regulated by regulator proteins and negative feedback mechanisms. Dysregulation of this pathway could induce a wide array of diseases including cancer. Oncogenic mutations in various components of the RTK/RAS/RAF/MEK/ERK pathway have been found in various cancers, with mutations of RAS and RAF being the most common in all cancer types.

Under normal cellular homeostasis, the Ras protein is cycling through GDP state (inactivation) and GTP state (activation) depending on the external stimuli or its interacting proteins. Oncogenic RAS mutations enable mutant Ras protein to be constitutively activated and continuously turned “on” leading to hyperphosphorylation and hyperactivation of the RAS/RAF/MEK/ERK pathway that contributes to the pathogenesis of several cancer types. Since the discovery of RAS as transforming oncogenes 40 years ago, there has been continuously ongoing interest in Ras research. An interesting finding from recent studies revealed a novel pathological role of argonaute 2 (AGO2), a Ras interacting protein, in promoting tumor progression in multiple mouse models of KRAS-driven pancreatic cancer and non–small cell lung cancer (Shankar et al., 2020; Tien et al., 2021). KRAS-AGO2 interaction is critical for PDAC progression, whereas disruption of this interaction by phosphorylation of AGO2 prevents progression to invasive tumor (Shankar et al., 2020; Tien et al., 2021).

Similarly, oncogenic mutation of RAF (mostly BRAF V600E mutation) induces constitutional activation of RAF kinase without stimulation, thereby leading to hyperactivation of the downstream signaling cascade. Alterations in this pathway are widespread across all tumor types. In an analysis of over 9000 TCGA tumors, the RAS/RAF/MEK/ERK pathway was the most frequently genetically altered pathway among the oncogenic signaling pathways, affecting about 46% of all the tumor samples analyzed (Sanchez-Vega et al., 2018).

Upstream of Ras are membrane receptors that initiate the signal transduction upon activation by extracellular stimuli. As the major membrane receptors of the MAPK signaling, tyrosine kinases (RTKs) undergo autophosphorylation after engagement of growth factor ligands and subsequently activate downstream signaling proteins to propagate signaling pathways. Mutations leading to constitutive activation of RTKs in the absence of upstream stimuli may thus induce oncogenesis. For example, epidermal growth factor receptor (EGFR) gene mutations predominantly occur in its tyrosine kinase domain, leading to the mutant EGFR protein staying at constitutive phosphorylated stage, thereby activating its downstream signaling and conferring oncogenic properties. In a recent large scale proteogenomic analysis of lung cancer tissues, several ALK driver chromosomal rearrangements and missense mutations of STK11 were identified (Gillette et al., 2020). ALK is a RTK that activate multiple downstream signaling cascades, including RAS/MAPK and PI3K, to promote cell proliferation and survival. Oncogenic fusion protein EML4-ALK resulting from chromosomal rearrangements are constitutively activated and can directly phosphorylate its downstream proteins without ligand binding to induce oncogenesis. STK11 is an intracellular kinase that functions as a tumor suppressor by phosphorylating and activating AMP-activated protein kinase (AMPK) to maintain cell polarity and inhibit cell proliferation and energy metabolism. Missense mutations in STK11 inactivate the kinase activity of STK11 and abolish its tumor suppressor activity, thereby contributing to tumorigenesis(Li et al., 2015).

2.2. Wnt-β-catenin pathway.

Wnt signaling pathway, a key cascade regulating cell stemness and cell-fate specification, is tightly activated and deactivated by phosphorylation and other PTMs. In the resting cell, the cytosolic β-catenin is deactivated through phosphorylation by a multi-protein destructing complex, and further degraded by ubiquitination. When Wnt ligands bind to the surface receptor Frizzled (FZD) and co- receptor low-density lipoprotein receptor-related protein (LRP), this trimeric complex then triggers phosphorylation of LRP, which further phosphorylates and recruits the components of the β-catenin destructing complex, leading to dissociation of the destruction complex. As a result, the phosphorylation of β-catenin is blocked, and β-catenin is then stabilized and accumulated, which then enters the nucleus where it promotes the transcription of Wnt target genes (Bugter et al., 2021). In this signaling cascade, phosphorylation plays the primary force in both activating LRP at Wnt-on state, and inactivating β-catenin at Wnt-off state. Wnt pathway is subject to numerous negative feedback regulations to maintain balanced signaling. Mutations causing sustained Wnt–β-catenin activation occur in most major cancer types, being most prominent in colorectal cancer, where over 90% colorectal cancers display mutations in this pathway (Sanchez-Vega et al., 2018).

2.3. Cell cycle pathway.

Cell cycle progression is regulated through a series of cell cycle regulators, whose expression levels are regulated at the post-translational level, mainly through Cyclin-dependent kinases (CDKs) mediated phosphorylation or ubiquitination (Liu et al., 2021). When there is a mitogenic signal, cell cycle progression is initiated through phosphorylation of Rb proteins by CDKs, which then release transcriptional factor E2F to promote transcription of downstream targets, leading to transition from G1 to S phase. When there are DNA double-strand breaks, protein kinase ATM (ataxia telangiectasia mutated) is recruited to phosphorylate several cell cycle regulators which then activate the DNA damage checkpoint, leading to cell cycle arrest, DNA repair or apoptosis. Dysregulation in cell cycle pathway, especially in the cell cycle checkpoints, is one of the major mechanisms for cancer cells to sustain uncontrolled proliferative signaling. Up to 45% of the over 9000 TCGA tumors had alterations in cell cycle pathway, only next to the RAS pathway which had the highest alteration frequency (Sanchez-Vega et al., 2018).

2.4. Other pathways relevant to hallmarks of cancer.

Phosphorylation is a very common modification of proteins that can rapidly switch proteins between activated and inactivated states. In the study analyzing the oncogenic signaling pathway of over 9000 tumor samples (Sanchez-Vega et al., 2018), the top ten most frequently altered pathways include RTK/RAS, cell cycle, PI3K, p53, Notch, Wnt, Myc, Hippo, TGFβ, and Nrf2 pathways. In addition to Ras, Wnt, and cell cycle pathways, almost all other frequently altered oncogenic signaling pathways found in cancers also involve changes in phosphorylation of key proteins, affecting their stability and activity (Sanchez-Vega et al., 2018).

2.5. Targeting phosphorylation as a cancer therapeutic strategy.

For nearly four decades, Ras was thought to be an “undruggable” cancer target. Although there are some recent breakthrough targeting a specific mutation (KRASG12C), no clinically effective therapies directly targeting Ras for RAS-mutant cancers have been developed yet (Stalnecker and Der, 2020). It is now clear that Ras proteins are also regulated by a vast array PTMs. Phosphorylation at several residues of Ras proteins have recently been shown to regulate Ras activities (Campbell and Philips, 2021). Currently, several small molecule inhibitors targeting various Ras phosphorylation sites are under preclinical studies or clinical trials (Campbell and Philips, 2021) (Table 1). Due to the critical role of RTKs in cancer development, substantial interests have been on targeting oncogenic mutations of RTKs for cancer therapeutics. Today, there are many FDA approved targeted therapies on RTKs (e.g. EGFR, HER2/HER3, ALK), using either RTK inhibitors or monoclonal antibody to combat the phosphorylation and constitutive activation of RTKs (Table 1). Inhibitors targeting other members of the MAPK cascade are also in various development stages, including preclinical and clinical studies, while some are already FDA approved (Table 1) (Yip and Papa, 2021).

Table 1.

Selected drugs or compounds trageting PTMs.

Gene/protein Target	Targeted Modification	Drugs	Cancer	Phase
20S proteasome	Ubiquitination	Bortezomib, Carfilzomib,	Myeloma, Lymphoma, Lung, Pancreas	FDA approved
ALK	Phosphorylation	Alectinib, Brigatinib, Ceritinib, Crizotinib, Lorlatinib	Lung	FDA approved
BRAF	Phosphorylation	Dabrafenib, Encorafenib, Vemurafenib	Melanoma, Lung, Colon, Rectum	FDA approved
CDK4/6	Phosphorylation	Abemaciclib, Ribociclib, and Palbociclib	Breast	FDA approved
CRBN	Ubiquitination	Thalidomide, Lenalidomide, Pomalidomide	Myeloma	FDA approved
EGFR	Phosphorylation	Afatinib, Almonertinib, Dacomitinib, Erlotinib, Gefitinib, Osimertinib, Amivantamab, Mobocertinib, Cetuximab, Panitumumab	Lung, Head and neck, Colon, Rectum	FDA approved
HDAC	Acetylation	Vorinostat, Belinostat, Panobinostat, and Chidamide, Romidepsin	Lymphoma, Myeloma	FDA approved
HER2/HER3	Phosphorylation	Lapatinib, Neratinib, Tucatinib, Trastuzumab and Pertuzumab	Lung, Breast	FDA approved
Mdm2	Ubiquitination	PRIMA	Liver, Pancreas	FDA approved
MEK1/2	Phosphorylation	Binimetinib, Trametinib, and Cobimetinib	Melanoma, Lung	FDA approved
PD-L1	Glycosylation	gPD-L1	Breast	Preclinical
PI3K	Phosphorylation	Copanlisib	Lymphomas	FDA approved
PI3K	Phosphorylation	Copanlisib	Breast	Phase II
Ras/SHP2	Phosphorylation	TNO155, JAB-3068, RMC-4630	Solid tumors	Phase 1, 2a
Wnt/LRP6	Phosphorylation	Salinomycin, Rottlerin, and Monensin		Preclinical
Wnt/SRSF	Phosphorylation	SM08502	Solid tumors	Phase 1
Wnt/β-catenin	Ubiquitination	MSAB	Colon	Preclinical

For cell cycle and DNA damage repair pathways, inhibitors targeting CDK4/6 activation have made significant progresses in breast cancer treatment, with three FDA-approved CDK4/6 inhibitors in clinics (Table 1) (Yip and Papa, 2021). Due to the critical role of WNT–β-catenin signaling in cancer development, especially in colorectal cancer, substantial efforts have been directed in developing therapeutics targeting this pathway. Numerous Wnt pathway antagonists, monoclonal antibodies and small molecule inhibitors have been developed, although none of these drugs has been clinically approved yet (Table 1) (Jung and Park, 2020). On-target toxicity is a common challenge in blockage of Wnt pathway that needs to be addressed in the development of Wnt targeting therapy.

The success of recent development in targeted therapies has revolutionized the treatment of cancer patients and indicated a new era of precision medicine (Du and Lovly, 2018). Despite these exciting advancements, inherent limitations on drug toxicity and drug resistance still cause treatment failure. Novel approaches have been shown to effectively overcome acquired resistance, including the development of next-generation drugs and combinational use of therapy (Du and Lovly, 2018).

3. Acetylation

Lysine acetylation is a ubiquitous PTM that extensively presents across the entire proteome, contributing to the regulation of a wide range of cellular functions for maintaining cellular homeostasis. More than 35,000 acetylation sites have been reported to date (Choudhary et al., 2009; Gil et al., 2017; Kim et al., 2006; Lundby et al., 2012; Zhao et al., 2010), providing an enormous functional diversity that may be implicated in the tumorigenesis or the manifestations of malignancies (Ali et al., 2018; Hu et al., 2022; Liu et al., 2017; Shvedunova and Akhtar, 2022; Wang and Zhao, 2022). We attempted to exemplify the complex biology of acetylation in cancer by overviewing its implications in histone regulation, metabolic pathways, oncoproteins and tumor suppressors.

As one of the major epigenetics systems, histone proteins can have various PTMs, including acetylation, methylation, ubiquitination, phosphorylation, and SUMOylation, all of which can affect the chromatin conformation and subsequent gene expression. Among these PTMs, alterations in histone acetylation play important roles in cancer development and progress (Fullgrabe et al., 2011; Harachi et al., 2021). Histone acetylation may occur on all core histone proteins. The well-studied histone H4 acetylation on lysine 16 (H4K16ac) is usually localized at the promoters and enhancers of genes that its acetylation state can activate gene transcription. Removal of the H4K16ac mark could decouple the binding of transcriptional factors at the promoters and subsequently inactivate gene transcription. Furthermore, loss of H4K16ac might also affect chromatin conformation and contribute to genome instability. Studies have evidenced that reduction in H4K16ac is associated with various cancer types and chemoresistance of cancer cells, and correlated with tumor progression (Elsheikh et al., 2009; Hajji et al., 2010). The global reduction of H4K16ac, together with loss of trimethylation of histone H4 at lysine 20 (H4K20me3) and DNA hypomethylation at repetitive DNA sequences are often considered as a common hallmark of malignant transformation (Fraga et al., 2005). In breast cancer, low or absent H4K16ac might be an early sign of cancer development, while moderate to low levels of H3K9ac, H3K18ac and H4K12ac were associated with poorer prognostic subtypes (Elsheikh et al., 2009). Downregulations of H3K9ac, H3K18ac and H4K16ac were strongly correlated with the risk of prostate cancer recurrence (Seligson et al., 2005). In the case of non-small cell lung carcinoma (NSCLC), upregulation of H4K5ac and H4K8ac and downregulation of H4K12ac, H4K16ac and H2AK5ac were correlated with the progression of NSCLC (Barlesi et al., 2007; Van Den Broeck et al., 2008), while reduction in H3K9ac was associated with better survival (Barlesi et al., 2007).

Since histone acetylation directly impacts gene transcription and is involved in the process of tumorigenesis, small molecules targeting histone acetylation are being developed as potential anti-cancer therapeutic drugs. Currently, there are five FDA approved histone deacetylase (HDAC) inhibitors for cancer treatment, with many more inhibitors targeting histone acetylation are still under clinical trials (Table 1) (Wu et al., 2020).

Lysine acetylation of non-histone substrates, including many oncoproteins, tumor suppressors and important enzymes involved in cancer metabolism, is significantly implicated in tumorigenesis (Harachi et al., 2021; Kaypee et al., 2016). The regulatory effects of acetylation on these proteins are multifaceted. For example, by competing lysine residues with ubiquitination, which mediates proteins for proteasomal degradation, acetylation can influence protein stability of p53, SMAD7 and c-MYC (Gronroos et al., 2002; Ito et al., 2002; Patel et al., 2004). Acetylation can also influence protein-protein interactions (Cohen et al., 2004) and subcellular localization of proteins (di Bari et al., 2006). Mutations of KRAS are common for the development of many cancers. A study showed that acetylation of KRAS at K104 attenuated its transforming activity and was a negative regulatory modification (Yang et al., 2012). For tumor suppressor p53, acetylation at K305, K370, K372, K373, K381, K382 and K320 increases its DNA-binding ability and activates its transcription target genes (Glozak and Seto, 2007).

Acetylation also influences the function of many key proteins in the regulation of tumor metabolism. Up to 20% of mitochondrial proteins have acetylation that may affect numerous metabolic pathways, such as the TCA cycle, urea cycle, and oxidative phosphorylation (Harachi et al., 2021). Alterations in the mitochondrial acetylation could contribute to the aerobic glycolysis and other metabolic reprogramming phenomenon observed in cancer. For example, acetylation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an essential enzyme in glycolysis, at K254 was found to accelerate tumor growth (Li et al., 2014). The reduction in acetylation of lactate dehydrogenase (ALDH-A) at K5 during the initiation of pancreatic cancer was found to promote tumor metabolism and cancer progression (Zhao et al., 2013).

4. Glycosylation

Most secretory and membrane-bound proteins produced by mammalian cells contain covalently linked glycans with diverse structures (Rudd and Dwek, 1997). Glycosylation plays a pivotal role in many biological processes, such as protein folding, cell adhesion and trafficking, cell signaling, pathogen recognition and immune response (Haltiwanger and Lowe, 2004; Helenius and Aebi, 2001; Kudelka et al., 2020; Laubli and Borsig, 2019; Marth and Grewal, 2008; Ohtsubo and Marth, 2006; Peixoto et al., 2019; Rudd et al., 2001; Woods et al., 1994). The most common forms of glycosylation are N-linked and O-linked glycosylation (Bertozzi and Kiessling, 2001; Khoury et al., 2011; Varki et al., 2015). While O-linked glycans are linked to the hydroxyl group on serine (Ser) or threonine (Thr) residues, N-linked glycans are attached to the amide group of asparagine residues in a consensus Asn-X-Ser/Thr sequence (X can be any amino acid except proline) (Bause, 1983; Bertozzi and Kiessling, 2001; Khoury et al., 2011; Varki et al., 2015). Glycan synthesis is not template bound but involves the concerted action of glycosidases, glycosyltransferases, and glycan-modifying enzymes. Environmental factors, immune pressure and altered metabolic mechanisms can lead to genetic and epigenetic modification of these enzymes and result in altered glycan biosynthesis and protein glycosylation that drive several key biological processes in cancer (Kannagi et al., 2010; Pinho and Reis, 2015). The known aberrant glycosylation implicated in cancer may include changes in global sialylation and fucosylation, N- and O-linked glycan branching, O-glycan truncation (Arnold et al., 2008; Christiansen et al., 2014; Hakomori, 2002), as well as abnormal glycosylation occupancy (Pan et al., 2014).

The modifications of carbohydrates may be cell-, protein- and glycosylation site-specific, and involve incomplete synthesis or neo-synthesis of glycans (Hakomori, 2002; Hakomori and Kannagi, 1983). Increased sialylation, such as sialyl Lewis a (SLe^a) and SLe^x), have been associated with many cancers (Kannagi et al., 2008). While SLe^a is the antigen of CA19–9 clinical assay for pancreatic and other GI-tract cancers for therapeutic monitoring (Indellicato et al., 2020; Luo et al., 2021), the expression levels of SLe^x, which is a ligand for selectins (Rosen and Bertozzi, 1994), have been correlated with poor cancer survival (Amado et al., 1998; Baldus et al., 1998). Core fucosylation is the addition of α1,6-fucose to the innermost GlcNAc residue of N-glycans. Increase of core fucosylation through the action of Fuc-TVIII have been associated with multiple cancers, including breast, lung and liver cancer (Hutchinson et al., 1991; Liu et al., 2011; Potapenko et al., 2010). Core fucosylated α-fetoprotein (AFP-L3) is a clinical biomarker for the early detection of hepatocellular carcinoma (HCC) (Aoyagi et al., 1985). The increased expression of complex β1,6-branched N-linked glycan are frequently associated with malignant transformation (Dennis et al., 1987). The abnormal expression of shortened or truncated O-linked glycan, including disaccharide Thomsen–Friedenreich antigen (T antigen) and the monosaccharide GalNAc (Tn), as well as their sialylated forms (ST and STn, respectively), have been associated with many cancers (Pinho et al., 2007; Radhakrishnan et al., 2014).

In addition to their direct implication in malignancy, glycans also profoundly influence protein functionality (Helenius and Aebi, 2001), altering many biological processes in cancer, such as inflammation, immune surveillance, cell adhesion, extracellular modification, inter- and intracellular signaling, and cellular metabolism (Pinho and Reis, 2015). Proteomic study and network analysis have found a group of cancer associated glycoproteins, including MUC5AC, CEACAM5, IGFBP3 and LGALS3BP, substantially hyper-glycosylated in pancreatic ductal adenocarcinoma tissue, and their increased activity of N-glycosylation was implicated in TGF-β, TNF and NF-kappa-B pathways (Pan et al., 2014). Mucins are a heavily glycosylated protein family that is well known to be associated with epithelial cancers, such as colorectal and pancreatic cancers. The glycan component can make up more than 50% of the molecular weight of a mucin glycoprotein, modulating their functionality in tumorigenesis as well as cancer cell interaction with the tumor microenvironment (Andrianifahanana et al., 2006; Chugh et al., 2015; Kudelka et al., 2015; Nagata et al., 2007; Pan et al., 2016). E-cadherin is a tumor suppressor protein and a biomarker for the epithelial mesenchymal transition (EMT). The addition of GnT-V-mediated β1,6GlcNAc-branched N-glycans to E-cadherin compromises cell-cell adhesion, promoting cancer invasiveness and metastases (Guo et al., 2003; Pinho et al., 2013). In cancer metabolism and signaling, increased O-GlcNAcylation crosstalk with phosphorylation can stabilize c-MYC, a cancer suppressor protein, contributing to oncogenesis (Itkonen et al., 2013).

The majority of the cell-surface receptors are transmembrane glycoproteins, such as epithelial growth factor receptor (EGFR), IGF receptor (IGFR), fibroblast growth factor receptor (FGFR), TGF β receptor (TGFβR) and integrins. Increased complex N-glycan number and branching affects the stability and retention of receptors at cell surface for proper functioning by modulating the interaction of branched N-glycans with galectin-3 and galectin-1, thus altering the signaling (Lau et al., 2007). In addition, aberrant glycosylation on cancer cell surface can modulate their interactions with various lectins, which mediate immune and inflammatory responses, and help tumor cells escape immune surveillance (Liu and Rabinovich, 2005).

Immunosuppressive ligand, programmed death-ligand 1 (PD-L1), is frequently overexpressed and heavily glycosylated in cancer tissues. Studies found that the glycosylation in PD-L1 was required for proper interaction with receptor programmed cell death protein-1 (PD-1) in triple-negative breast cancer (Hsu et al., 2018; Li et al., 2018). Antibody specifically targeting the glycosylated PD-L1 was able to block PD-L1/PD-1 interaction and promote PD-L1 internalization and degradation, providing a potential approach targeting protein glycosylation to enhance immune checkpoint therapy (Li et al., 2018).

Given the pivotal role of protein glycosylation in many biological processes and the tendency of glycoprotein entering circulation, malignancy associated glycoproteins represent promising biomarkers for diagnostics and prognostics. In fact, many currently FDA-approved cancer biomarkers are glycoproteins or glycosylation assays, including: a-fetoprotein (AFP) in liver cancer; prostate-specific antigen (PSA) in prostate cancer; cancer antigen 125 (CA125) and human epididymis protein 4 (HE4) in ovarian cancer; carcinogenic embryonic antigen (CEA) in colorectal cancer, CA 19–9 in pancreatic cancer, HER2/NEU and CA15–3 and CA27–29 in breast cancer, and thyroglobulin (Tg) in thyroid cancer (Kirwan et al., 2015).

5. Palmitoylation

Protein lipidation is the covalent addition of various lipids to the residues of proteins, including cysteine, serine, lysine, or histidine (Nadolski and Linder, 2007). Protein lipidation increases the hydrophobicity of target proteins and affects their conformation and stability, binding affinity to membranes, subcellular localizations, and association with other biomolecules. Therefore, lipidation can regulate protein function, and is mechanistically linked to lipid metabolism and cellular energy homeostasis. Among the diverse types of lipidation, palmitoylation is the most common and best studied lipid modification (Linder and Deschenes, 2007). Protein palmitoylation has been associated with various cancer, and aberrantly palmitoylated proteins may indicate their functional changes in cancer (Chen et al., 2018; Chen et al., 2020; Yang et al., 2010). In addition, fatty acid synthase, the enzyme system which catalyzes palmitate synthesis, is frequently upregulated in cancers and has been identified as a potential therapeutic target (Fhu and Ali, 2020; Menendez and Lupu, 2007).

Palmitoylation as well as depalmitoylation is implicated in cancer metabolism and the physiological state of mitochondria. Palmitoylation of CD36 increases fatty acid uptake and oxidation, facilitating liver cancer development (Zhao et al., 2018). Palmitoylation of estrogen receptor α and GLUT4 increases glucose uptake in estrogen receptor-positive breast cancer cells and CHO-IR cells, respectively (Garrido et al., 2013; Ren et al., 2015). Palmitoylation of TMX1 and CKAP4 increases mitochondrial respiration (Harada et al., 2020; Raturi et al., 2016). Reversely, depalmitoylation of KRAS4A (Amendola et al., 2019) and PRDX5 (Cao et al., 2019) increases glycolytic flux and mitochondrial redox buffering capacity, respectively. These studies demonstrated that palmitoylation, as well as other lipidations, play a crucial role in impacting cancer metabolism. In cancer development, protein lipidation is a potent regulator of apoptotic calcium signaling (Chen and Boehning, 2017). Studies have also suggested that lipidation of Wnt proteins, which regulates cell proliferation and differentiation, are implicated in intestinal carcinogenesis and cancer (Kaemmerer and Gassler, 2016). Some nonspecific inhibitors of protein palmitoylation have been developed for research. However, at present, no therapeutic drugs have been developed to target the palmitoylation status of specific target proteins, such as Ras proteins (Cox et al., 2015).

6. Ubiquitination and SUMOylation

Ubiquitination is the addition of the evolutionally conserved ubiquitin protein to target proteins (Hershko and Ciechanover, 1998). The multistep process is mediated by ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). In addition to its important role in proteasome degradation, the physiological functions of ubiquitination also involve non-proteolytic signaling, such as regulation of inflammatory pathway, autophagy, DNA repair, T cell receptor signaling and enzymatic activity (Bhattacharjee and Nandi, 2017; Kattah et al., 2017; Martin-Vicente et al., 2017). In cancer, ubiquitination plays a pivotal role in tumorigenic pathways by regulating the activity or degradation of tumor promoting or suppressor proteins. CDKs are the primary drivers of cell cycle transition and its activities are regulated through ubiquitination of the key regulators, including cyclins, CDK inhibitors (CKIs), other kinases and phosphatases (Nakayama and Nakayama, 2006). Deregulation of ubiquitination-proteasome system (UPS), such as ubiquitination status of cyclins, might result in uncontrolled proliferation, migration and genomic instability in cancer (Dong et al., 2018; Liu et al., 2019; Shan et al., 2009). The dynamic and reversible ubiquitination of tumor suppressor p53 is a central mechanism that controls p53 regulation. In cancer cells, p53 undergoes ubiquitination by interacting with the RING finger E3 ubiquitin protein ligase MDM2, and is subsequently subject to degradation by UPS (Kussie et al., 1996). In a cancer-associated inflammatory response, ubiquitination is utilized to regulate various components of the TNF-induced signaling complexes by ubiquitin conjugation and deubiquitination. Ubiquitination of RIPK1, a key regulator of TNF-mediated apoptosis, necroptosis and inflammatory pathways, on lysine 377 in human was shown to be critical for NF-kB activation (Li et al., 2006). In TGF-β signaling cascade, higher protein ubiquitination and accelerated degradation of downstream SMAD4 can lead to inhibition of transcriptional response of TGF-β (Wan et al., 2005).

Similar to ubiquitination, SUMOylation is the covalent attachment of small ubiquitin-like modifier (SUMO) to the lysine residues in target proteins (Seeler and Dejean, 2017). Known SUMO isoforms include SUMO1, SUMO2/3 and SUMO4. In cancer, many oncoproteins and tumor suppressors are functionally regulated via SUMOylation. For example, BRCA1 is a tumor suppress gene associated with a high risk of breast and ovarian cancer, and its activity is regulated by the SUMO pathway. BRCA1 is SUMOylated in response to genotoxic stress, and co-localizes at sites of DNA damage with SUMO1, SUMO2/3 and the SUMO-conjugating enzyme Ubc9 (Morris et al., 2009). SUMO pathway plays an important role in many aspects of carcinogenesis, including DNA damage response, cancer cell proliferation, invasion, metastasis and apoptosis.

Targeting the dysregulated UPS is an active area of therapeutic development against cancer. Many inhibitors have been developed targeting different components of the UPS, including the proteasome, E3, E1, E2, and DUBs (Deng et al., 2020). Currently, there are five FDA approved ubiquitination targeting drugs and many more are under preclinical studies and clinical trials for cancer treatment (Table 1) (Deng et al., 2020).

7. PTM crosstalk

PTM crosstalk or PTM code is the complex and dynamic interplay between multiple PTMs to influence the actions of each other (Huang et al., 2019; Lothrop et al., 2013; Minguez et al., 2015; Minguez et al., 2012; Wu et al., 2019). PTM crosstalk can occur in the fashion of either intraprotein or interprotein, between the same or different types of modifications. Regardless of the actions, PTM crosstalk can orchestrate sophisticated interactions of PTMs to influence the functions, signaling pathways and regulation of protein networks in tunorigenesis. For instance, Ras/MAPK, TGF-β/Smad, PD-L1/PD-1 and TP53 pathways are among the most important regulatory signaling pathways involved in various cancers and metastasis. In the RAS/MAPK pathway, KRAS and other signal mediators are subject to multiple PTMs, including phosphorylation, ubiquitination, farnesylation, proteolysis, methylation and palmitoylation (Ahearn et al., 2011; Laude and Prior, 2008). These PTMs regulate the trafficking and localization of KRAS into the membrane, and influence KRAS activity and signaling. Dysregulation of the TGF-β signaling pathway is commonly implicated at cancer initiation and progression. Many signaling mediators in the TGF-β pathway are subject to extensive PTMs (Xu et al., 2016), including phosphorylation and ubiquitination, which are critical for the initiation and regulation of the signal transduction into the nucleus. As discussed in above, the key T-cell checkpoint ligand, PD-L1, which shows increased positivity in multiple cancers (Teng et al., 2015; van der Woude et al., 2017), is subject to extensive regulation by PTMs, including phosphorylation,N-linked glycosylation, acetylation and ubiquitination (Hsu et al., 2018; Li et al., 2016). Aberrant alterations of PTMs directly affect PD-L1 protein stabilization, subcellular localization and PD-L1-mediated immune resistance (Hsu et al., 2018). The activation/inactivation of tumor suppressor p53 functions, which regulates cellular responses to various stress signals, is modulated by a wide spectrum of PTMs, including phosphorylation, ubiquitination, acetylation, methylation, SUMOylation and neddylation (Bode and Dong, 2004; Dai and Gu, 2010). These are a few examples among the numerous PTM crosstalks implicated in cancer. As an emerging field in PTM studies, PTM crosstalk in many diseases, including cancer, remains largely unexplored.

8. Mass spectrometry-based PTM analysis

Mass spectrometry (MS)-based proteomics is currently the best available tool to meet the challenges for proteome-wide PTM analysis, offering unprecedented resolution and throughput for precise site mapping and structure identification, qualitatively or quantitatively (Olsen and Mann, 2013). The compositional and structural modifications in various amino acid residues due to PTMs can be dramatically different and may require different strategies for analysis. Furthermore, the enormous complexity and vast dynamic range of protein abundances in a proteome pose additional challenges in PTM analysis, as many peptides with PTMs are low abundance and subject to low sensitivity for MS detection. Using phosphorylation, glycosylation, ubiquitination, acetylation and palmitoylation as examples, Figure 2 illustrates the generic workflow for a PTM characterization. The concerted approaches typically comprise of several integrated modules, including sample preparation, PTM enrichment, chemical/biological cleavage or derivatization, separation strategy and mass spectrometric acquisition, followed by corresponding bioinformatics analysis.

Fig. 2.

A generic workflow of integrated PTM analysis, using glycosylation, phosphorylation, ubiquitination, acetylation and palmitoylation as examples.

For many PTMs with small universal chemical groups, such as phosphorylation, and acetylation, tens of thousands of sites can now be precisely located and confidently identified in the sequence of the protein in complex biological samples (Gil et al., 2017; Jensen, 2004; Ke et al., 2016; Low et al., 2020; Pagel et al., 2015; Paulo and Schweppe, 2021; Polat and Ozlu, 2014; Riley and Coon, 2016; Roux and Thibault, 2013; Simithy et al., 2018; Trost et al., 2010; Zhao and Jensen, 2009). Nonetheless, a comprehensive, quantitative analysis of these PTMs has never been trivial, and may involve multiple steps of optimizations in purification, enrichment and quantification prior to MS analysis. For instance, phosphoproteomics may be enhanced by strategies for phosphoprotein or phosphopeptide enrichment, such as IMAC, TiO₂ or HILIC, as well as MS technique of soft fragmentation, such as electron-transfer dissociation (ETD) (Junger and Aebersold, 2014; Riley et al., 2017).

For PTMs with complex and heterogeneous structures, such as glycosylation, ubiquitination, SUMOylation, lipidation, various strategies have been developed to overcome the analytical hurdles. Below we will briefly discuss several chemical or biological methods that are often utilized to proteolyze or derivatize these PTMs to generate specific mass tags that can be recognized by MS analysis.

A glycoproteomic analysis is complicated not only by the variety of carbohydrates, but also by the complex glycosidic linkage of the glycan to the protein. Various methods have been developed for enrichment of glycoproteins or glycopeptides to enhance a glycoproteomic analysis, such as lectin affinity, hydrazide chemistry, hydrophilic interaction and size-exclusion (Alagesan et al., 2020; Narimatsu et al., 2018; Pan et al., 2011; Peng et al., 2021; Riley et al., 2021). Conventional technologies can separately profile the deglycosylated glycoproteins using a proteomic approach (Liu et al., 2013; Pan et al., 2011; Sjostrom et al., 2015; Wollscheid et al., 2009; Zhang et al., 2003; Zhang et al., 2007), or identify the cleaved glycan structures using glycomics (Morelle and Michalski, 2005; Orlando, 2010; Shriver et al., 2004; Vercoutter-Edouart et al., 2008; Wuhrer, 2013; Wuhrer et al., 2009). However, a technical hurdle still exists in integrating the information together to define the glycoforms with site-specific glycan structures. With the recent advances in MS and bioinformatics, hybrid fragmentation technologies, such as stepped higher energy collisional dissociation (HCD) or electron-transfer/higher energy collision dissociation (EThcD), have been increasingly applied for intact glycopeptide analysis to obtain in situ information on site-specific glycoforms (Bollineni et al., 2018; Brunner et al., 2015; Gaunitz et al., 2017; Hoffmann et al., 2016; Riley and Coon, 2018; Thaysen-Andersen et al., 2016; Ye et al., 2019; Zhu et al., 2020).

The analysis of ubiquitination is enhanced by targeting the diglycyllysine Lys-ε-Gly-Gly (K-ε-GG) remnant produced by tryptic digestion of proteins, which cleaves ubiquitinated lysine side-chains (Kang and Yi, 2011; Udeshi et al., 2013). The K-ε-GG remnants in ubiquitinated lysine residues not only serve as a mass tag for precise mapping of ubiquitination site, but also can be used for enrichment of ubiquitinated peptides using antibodies that recognize K-ε-GG. In SUMOylation analysis, tryptic digestion of small ubiquitin-like polypeptides produce large branched peptides instead of K-ε-GG. These large remnants of SUMO conjugates drastically complicate the fragmentation pattern of the SUMOylated peptides, hampering the identification of SUMO acceptor sites in target proteins (Filosa et al., 2013; Hendriks et al., 2014; Lamoliatte et al., 2014; Tammsalu et al., 2015). A recent effort has demonstrated a method for proteome-wide detection of endogenous SUMOylation using α-lytic protease, which digests SUMO conjugates and generates SUMO-remnant K-ε-GG for MS analysis (Lumpkin et al., 2017).

Methods have also been developed to enrich and characterize protein palmitoylation, including hydroxylamine-mediated acyl-biotin exchange (ABE) and click chemistry based approaches (Collins et al., 2017; Drisdel and Green, 2004; Forrester et al., 2011; Hang et al., 2007; Martin and Cravatt, 2009; Yang et al., 2010). Using combinations of these approaches enabled MS identification of many palmitoylated proteins in various biological samples (Collins et al., 2017; Rodenburg et al., 2017; Thinon et al., 2018; Wang and Schey, 2018; Won and Martin, 2018).

Proteome-wide analysis of protein PTMs has become a unique domain in MS-based proteomics. Quantitative assessment of PTM status, and precise identification of PTM sites and detailed structures, especially those with complex and heterogeneous structures, still remain challenging and require integrated approaches that involve multiple technical aspects. In the human proteome, while a complete inventory of modification sites has not been established for any PTM, methods and strategies have been developed for many PTMs, and hundreds and thousands of PTM sites have been identified. The analysis of PTMs has emerged as a vibrant field in cancer research.

Conclusion Remarks

The implications of various PTMs in tumorigenesis, metastasis, and anti-cancer strategies have been increasingly recognized. Altered status of many PTMs are associated with cancer hallmarks and manifestations. This is an important emerging area in cancer research, which could lead to novel mechanisms and therapeutic strategies to facilitate early detection and treatment of cancer. Mounting efforts have been driven towards investigating dynamic occupancy or stoichiometry of site-specific PTMs and their interplays to establish PTM landscapes implicated in cancer. Given that PTMs afford another layer of regulation for the core proteins, exploiting PTMs for development of diagnostic markers and therapeutic targets is intriguing and has shown to be successful. It is expected that the advances in mass spectrometry and bioinformatics, as well as perspectives from systems biology, will facilitate PTM studies in cancer biology and clinical applications.