Post-translational Modifications (PTMs), from a Cancer Perspective: An Overview

Studies on post-translational modifications (PTMs) have grabbed attention of the scientific community worldwide, its role in pathogenesis of cancer and prognostic biomarkers associated with cancers. However, unraveling the specific role of PTMs in carcinogenesis or in predictive biomarkers requires holistic understanding of the cancer types and associated mechanisms. Manifestation of cancer is complex and involves multiple steps including modifications at the levels of genes, associated proteins and signaling pathways. Biomarkers, as a prognostic marker, are critical in deciding efficacy of the clinical outcomes in malignancies. Growing evidence suggests that several biomarkers that are post-translationally modified play important role in human cancers. In the current review, few of such biomarkers and targets that are post-translationally modified and are associated with carcinogenesis are collated and analyzed to provide a bird’s eye view of their role in cancer types. Such analysis will help in understanding the pathogenesis and the precise role of biomarkers in designing better therapeutic interventions for different cancer types.

Evolution of these technologies has offered ample potential in the hunt for molecular markers of early stage cancers.
However, these methods can be insufficient to investigate the dynamic nature of signaling processes that cells exhibit during their transformation to become tumor [3].
Acetylation has many important effects on oncoprotein p53, it increases p53 protein stability. In many cell types, inhibition of HDACs that remove acetyl groups from p53 (i.e., HDAC1 and SIRT1) causes increased p53 acetylation and p53-dependent activation of apoptosis and senescence [41]. Acetylation is an essential regulator of the anti-cancer functions of p53 [42]. In a study it was deduced that temporary and reversible inhibition of p53 acetylation in cancer subjects, especially those with p53-mutant tumors, may protect them from severe chemotoxicity [7].
It has been shown that expression of NATs may both be increased and decreased in cancer versus non-cancer tissues and these acetyltransferase enzymes have been suggested to act as oncoproteins as well as tumor suppressors in human cancers [37]. Acetylation is an important modification of proteins with effects on the metabolome level and is an important research area to understand its physiological consequences.

Methylation
Methylation is a comparatively budding and promising area of research which has emerged as a prevalent post-translational modification. Methylation of arginine/lysine residues on nonhistone proteins frequently mediates the transduction of cellular signals. With recent technological advancements the stage is now set to decode the 'methylproteome' to delineate its functions in health and disease [43].
Methylation of non-histone protein has an important regulatory role to play in a wide range of cellular processes [44] including transcriptional regulation, RNA metabolism and DNA damage repair. Various proteins involved in DNA repair (MRE11, p53, DNA polymerase b) have been shown to be controlled by arginine/lysine methylation [45].
With growing number of lysine methyltransferases (methylate non-histone proteins on lysine residue), a number of proteins like ERα, NF-κB, pCAF and other transcription factors have been identified that have implications in tumorigenesis and other metabolic disorders [1]. In addition to regulating gene expression, protein modification by methylation also contributes to the regulation of protein stability [46]. In a recent study, it was highlighted that BTG3 (B-cell translocation gene 3), a candidate tumor suppressor, promotes methylation of CHK1 (checkpoint kinase 1), a vital checkpoint kinase essential to normal cellular functions [15]. CHK1 has also been shown to be methylated in response to ultraviolet-induced DNA damage [47]. Arginine/lysine residues methylation is dynamic and reversible which can be removed by demethylation enzymes [50].
Identification of mechanisms involved in protein methylation/ demethylation remains an interesting area of research to understand the regulatory roles of target proteins in cancer and other diseases.

Phosphorylation
Phosphorylation is one of the most prominent PTM of proteins and is an important cellular regulatory mechanism as through phosphorylation and dephosphorylation events many proteins, enzymes and receptors are regulated.
Phosphorylation is a reversible mechanism and regulates numerous cellular processes such as protein synthesis, cell division, signal transduction, cell growth, development and aging which happen through protein kinases and phosphatases. Especially, the protein kinases are accountable for cellular transduction signaling and their overexpression or malfunction is found in several diseases, mostly tumors [51].
Therefore, targeting protein kinases using kinase inhibitors can be valuable for the treatment of cancer. The most common amino acid residues modified for phosphorylation are serine (Ser or S), threonine (Thr or T), and tyrosine (Tyr or Y), they also play indicative role in progression of cancer [18,52].
A number of cellular signalling pathways including tyrosine kinase, MAP kinase, cadherin-catenin complex and others are major players of the cell cycle, and deregulation in their phosphorylation-dephosphorylation cascade has been shown to be manifested in the form of various types of cancers [53]. Phosphorylation of Akt/protein kinase B (PKB), a serine/threonine kinase which mediates a variety of biological responses, has a prognostic and/or predictive role of in several cancers including breast, prostate and non-small cell lung cancer [54]. Dysregulated tyrosine kinase activity is reported in different types of cancers for example amplification of Her2/neu was observed in tumor cells of breast cancer [55].
MAPK cascade (SOS-Ras-Raf-MAP kinase pathway), involved in the regulation of normal cell proliferation, differentiation and apoptosis, has an important role in cancer growth and progression [56].
Additionally, the importance of Raf and MEK in cancer progression and in promoting cancer growth has been well established [57]. Tumor suppressor p53 phosphorylation sites, Ser315 and Ser392 in the C-terminal regulatory domain, are associated with elevated p53-dependent transcription [58]. To sustain the functional integrity of the cadherincatenin complex phosphorylation is an important process.
Dysregulation of this process has been found to be strongly associated with cell-adhesion defects in carcinomas including prostate cancer progression [53,59]. Studies have revealed that transforming growth factor β (TGF β) prevents the phosphorylation of retinoblastoma protein (pRb) which leads to its inactivation [60]. This signaling is altered in many human cancers [61], in some of which impassiveness of cyclin-CDK4 complex to the inhibitory signals of p15INK4B leads to inactivation of pRb by hyperphosphorylation [62].
Phosphorylation pathways are crucial regulators of normal cellular functioning and these pathways are needed to be considered for more rational treatment of cancer.

SUMOylation
SUMOylation is a widely occurring reversible PTM which has attracted increasing attention as it is involved in a number of biological processes for the maintenance of genomic integrity.

Ubiquitination
Ubiquitination, as a multistep, conserved and highly dynamic process, functions to degrade and recycle proteins. It is involved in additional cellular processes such as activation of NFkB inflammatory response and DNA damage repair [71]. Ubiquitination also affects the result of many lethal diseases like cancer [72]. Three main classes of ubiquitination enzymes are activating enzymes (E1), conjugating enzymes (E2), and ligases (E3). Unregulated expression of these enzymes, including DUBs (deubiquitinating enzymes) and other complexes (SCF complex) involved in ubiquitination mechanism, contributes to the signaling of various oncogenes leading to cancer progression and metastasis.
Ubiquitination displays parallel properties with phosphorylation but distinguishes itself in important ways [73]. As a marker, phosphorylation often triggers subsequent ubiquitination, in particular where ubiquitination leads to degradation [74], and in other cases ubiquitination provide a switching mechanism that can turn on/off the kinase activity of certain proteins [75].

PTMs based Cancer Markers
The and SOD3) and 61 hypo-methylated (e.g., HBE1, SNRPF, TPD52) markers for gallbladder cancer (GBC) [83]. Oncology drugs approved lately, target distinct cancer biomarkers or pathways in tumor cells. Table 2 lists some examples of the important cancer biomarkers, all of them are known to be post-translationally modified, and effectively targeted for the development of drugs. We have briefly discussed below some PTM based biomarkers associated with carcinogenesis.  [84]. Upon X-rays and chemotherapy treatments, EGFR becomes phosphorylated and this event is accompanied by receptor internalization provoking p38 or Src-dependent, and clathrin-and AP-2 adaptor-dependent endocytic trafficking [85,86,87]. It has been shown that abolishing p38-dependent EGFR internalization diminishes the efficacy of chemotherapy-induced cell death therefore promoting the cytotoxic effect of chemotherapy drugs such as Cisplatin [87]. Drugs, for example Gefitinib (Iressa) and Erlotinib (Tarceva), has been approved with either a companion or complementary diagnostic to target activating EGFR mutation (EGFR M+) in non-small cell lung cancer (NSCLC). Mutations involving the PTM sites of EGFR impair EGFR trafficking. EGFR sites, tyr1068 and tyr1173, are among the two most relevant phosphorylation sites of EGFR, and phosphorylation at tyr1068 has been identified as a powerful biomarker associated with strong Erlotinib sensitivity in lung cancer stem cells (LCSCs) [21]. In contrast, a recent study provided additional mechanistic aspects of EGFR regulation by showing that mutations preventing EGFR phosphorylation at tyr998 or in the ser1039 region abolished or greatly reduced EGFR interactions with Adaptin subunits AP-2 and AP-1, and resulted in impaired receptor trafficking [88]. Studying the biological effects of such events are is important to determine the efficacy of EGFR-targeting drugs.
Human epidermal growth factor receptor-2 (HER2) is another important cancer biomarker which is overexpressed in large number of breast cancers. HER2 is activated by phosphorylation at specific tyrosine residues. Strong expression of activated HER2 is associated with poor prognosis in HER2 positive breast cancer patients. Experiments on primary breast tumors showed that strong expression of HER2 phosphorylated at tyrosine 1221/1222 is associated with poor prognosis in HER2 positive breast cancer patients [89].
To treat HER2 positive breast cancer patients, drug Trastuzumab (Herceptin) is used which inhibits HER2 signaling and its subsequent activation. Inception of companion diagnostics was with Herceptin. Herceptin mechanism involves inhibition of HER2 cleavage and prevention of the production of an active truncated HER2 fragment [90]. However, Herceptin resistance has been noted in some patients possibly due to failure to abolish HER2 phosphorylation, therefore, alternate treatment opportunities are also being explored to overcome acquired resistance in breast cancers. Studies have confirmed that Herceptin although down-regulated HER2 receptors in HER2-positive breast cell lines but failed to decrease HER2 phosphorylation as this phosphorylation was maintained and increased by the ligand-induced activation of EGFR, HER3, and HER4 receptors, resulting in their dimerization with HER2, by a protein kinase B (PKB) negative feedback loop [91].
Similar feedback loops might also be involved in the acquired resistance to Herceptin, an area of further research.
Physical blockade of the HER2 receptor is a further proposed mechanism for Herceptin resistance, for example, the mucin molecule, MUC4, with its extended carbohydrate structure seems to function as a barrier for biomolecular interactions in the extracellular environment [92,93]. A recent study highlighted the importance of cellular glycosylation on the binding of the drug Herceptin to the surface of cancer cells, the responsiveness of cancer cells to a chemotherapeutic agent, and potential of glycosylation inhibitors as future combination treatments for breast cancer [94]. This study showed the importance of the glycocaylx in the accessibility of the HER2 epitope to Herceptin [94], influencing many aspects of cancer cell biology and drug responsiveness since glycosylation affects many proteins. Tn and its sialylated version STn carbohydrate antigens, expressed highly by many types of tumors, may serve not only as a prognostic marker but also as a therapeutic target [96]. Not only as a biomarker but also as a contributor to the cancer development, protein glycosylation is an important phenomenon. It is therefore imperative to explore tumorassociated glycosylation as it provides novel diagnostic and therapeutic targets.
Estrogen receptor α (ERα) is expressed in the majority of breast cancers and promotes estrogen-dependent cancer progression. Redox-and phosphorylation-based PTMs are important and common modifications of ERα along with several other reported ERα PTMs, many of these modifications modulate receptors activity in breast tumors [97]. Important phosphorylation sites have been identified in endogenous ERα derived from the human breast cancer cell lines [97].
Phosphorylation of ERα induced by a growth factor pathway might be one mechanism of enhanced activation of the estrogen signal [98], for example, sites targeted by kinases such as MAPK, Akt, and c-Src [99]. investigations is seen, however, with the emerging drugs, their resistance is also growing. This emerging resistance is another challenge scientists are weathering. Understanding resistance mechanisms will lead to deeper understanding of carcinogenesis and will also pave the way to discover new generation drugs with profound selectivity to overcome resistance. With new drugs, a large number of clinical trials will also be warranted to validate the efficacy of drugs.
Factual state of cancer progression may not be completely reflected by simply observing the changes in gene expression levels therefore it is important to study PTMs to find out the differences between normal and cancer tissues.