INTRODUCTION

Lung cancer is the leading cause of cancer-related deaths globally, with significant incidence and mortality rates. It accounts for approximately 2 million new diagnoses and 1.8 million deaths annually, making it the most common cause of cancer-related mortality worldwide.[1] Adenocarcinoma and squamous cell carcinoma are the two most common subtypes of non-small cell lung cancer (NSCLC), accounting for about 50% and 20-30% of lung cancer cases each year, respectively. Notably, the patient’s smoking history and histological subtype frequently correlate; for example, adenocarcinoma is more prevalent in patients with a history of never or light smoking. Furthermore, both types are linked to targetable genomic alterations.[2-4]

Over the past two decades, significant advancements in understanding the molecular biology of lung cancer have led to the identification of various oncogenic driver mutations that can be targeted with specific therapies. The majority of therapeutically significant oncogenic drivers are kinases that are activated by mutation, amplification, or by constitutively activating the kinase domain in fusion proteins. In NSCLC, the key receptor tyrosine kinases (RTKs) identified include epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER-2), cellular mesenchymal-epithelial transition factor (c-MET), rearranged during transfection (RET), anaplastic lymphoma kinase (ALK), and ROS proto-oncogene 1, receptor tyrosine kinase (ROS1). Oncogene addiction, the cancer cell’s dependence on specific oncogenes for survival, has led to the development of targeted treatments, primarily tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs), which attach to the active kinase domain’s ATP-binding pocket or block ligand-receptor binding, respectively.[5,6]

Although targeted treatments against RTKs have improved survival rates, resistance often develops, necessitating ongoing research into next-generation inhibitors and combination therapies to overcome this phenomenon. Post-translational modifications such as glycosylation of RTKs play a significant role in modulating signaling pathway activation status and are also associated with therapy resistance.[7] For example, inhibition of N-glycosylation disrupts RTK signaling in tumor cells.[8] This disruption can reduce RTK activity and radiosensitize tumor cells, suggesting a therapeutic approach to target primary and redundant RTK signaling.[7] This review highlights how glycosylation may interfere with the regulatory mechanisms of RTKs and modulate their ability to promote oncogenic signaling.

GLYCOSYLATION IN CANCER

Glycosylation is a critical post-translational modification involving the addition of carbohydrate chains, or glycans, to proteins and lipids. This process is essential for various physiological and pathological cellular functions by influencing cell signaling and cellular interactions.[9,10] In the context of cancer, glycosylation plays a significant role in tumor progression and immune modulation.[11,12] Changes in glycosylation may occur through genetic mutations or can be induced within the tumor microenvironment by factors such as hypoxia and inflammation that drive the activity of glycosyltransferases. Glycoproteins are proteins that have carbohydrate chains covalently attached to their polypeptide backbones, and these chains can constitute 1-80% of the total protein weight.[9,10] These structures are crucial for various biological functions, including cell-cell recognition and protein stability. Common sugars in glycoproteins include galactose, mannose, glucose, N-acetylglucosamine, and sialic acids.[10]

N- and O-linked glycans are the two types of glycosylation that occur in the endoplasmic reticulum (ER) and Golgi apparatus.[13,14] While N-glycosylation [Figure 1] always involves the same core glycan being attached to the amide nitrogen of asparagine (Asn) residue, O-glycosylation can take many different forms: Intracellular O-GlcNAc modification of nuclear and cytosolic proteins; O-xylose-linkage of glycosaminoglycans; O-mannosylation of dystroglycan, cadherins, and protocadherins; O-fucosylation and O-glucosylation of surface receptors; O-galactosylation of collagen; and the linkage of O-acetylgalactosamine (GalNAc) glycans, also known as mucin-type O-glycans.[13]

Figure 1: N-glycosylation of receptor tyrosine kinases. N-glycosylation begins in the endoplasmic reticulum (ER) with (1) assembly of an oligosaccharide precursor which is linked to the Asn residue of a protein. Correctly folded proteins are then transported from the ER to the Golgi apparatus through coat protein complex II (COP-II) vesicles, where (2) N-glycans are trimmed and extended by glycosyltransferases. Finally, proteins are transported (3) directly to the plasma membrane through transport vesicles where they can be activated through dimerization and ligand binding (4). ALGs: Mannosyltransferases, ALK: Anaplastic lymphoma kinase, c-MET: Cellular mesenchymal-epithelial transition factor, EGF: Epidermal growth factor, EGFR: Epidermal growth factor receptor, Fuc: Fucose, FUTs: Fucosyltransferases, GalTs: Galactosyltransferases, GalNAc: N-Acetylgalatosamine, GCSs: Glucosidases, GlnNAc: N-Acetylglucosamine, GnTs: N-acetylglucosaminyltransferases, G-rich: Glycine-rich, HER-2: Human epidermal growth factor receptor 2, LDL: Low-density lipoprotein class motif, MAM: Meprin, A5 protein and receptor protein tyrosine phosphatase mu domain, Man: Mannose, MNSs: Alpha-mannosidases, OSTs: Oligosaccharyltransferases, P: Protein-residue for post-translational modification, PSI: Plexin-semaphorin-integrin homology domain, SA: Sialic acid, ST6GAL1: ST6 beta-galactoside alpha-2,6-sialyltransferase 1, Created in BioRender. Meder, L. (2025) https://BioRender.com/s89u633.

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The main principle of O-glycosylation is that sugars are attached to the oxygen atom of serine (Ser) or threonine (Thr) residues in proteins. This process is tissue-specific and depends on the protein’s structure. The elongation and termination of O-glycans are regulated by specific transferases.[15] O-glycosylation has many functions, such as regulating immune responses, and it can modulate enzyme and signaling molecule activities by altering protein stability and facilitating functional recognition.[13] O-glycosylation is further regulated by exon shuffling and alternative splicing which can result in proteins differing in their presence or absence of Ser/Thr (/Proline)-rich domains.

Aberrant O-glycosylation is also linked to cancer metastasis. For instance, in pancreatic cancer, the loss of core1 β1,3-galactosyltransferase (C1GALT1) leads to immature O-glycosylation and truncation of CD44, resulting in Tn-antigen enrichment.[16] This, in turn, drives tumorigenesis, metastasis, and stemness properties. Little is known about the functional consequences of O-glycosylation alterations in lung cancer, but recent glycoproteomic studies have identified aberrantly glycosylated proteins in this disease.[17]

N-glycosylation is a highly conserved process and involves the attachment of an oligosaccharide precursor to the Asn residue of a protein within the consensus sequence Asn-X-Ser/Thr (NXS/T), where X represents any amino acid except proline. This NXS/T motif is essential for the addition of glycans and is commonly found in extracellular and luminal regions of glycoproteins.[18] The mechanism of N-glycosylation begins with the synthesis of a lipid-linked oligosaccharide precursor on the ER membrane, which is subsequently transferred altogether as a group to the nascent polypeptide chain by the enzyme oligosaccharyltransferase (OST). Once the oligosaccharide is attached, it undergoes a series of modifications, including trimming and processing by glycosidases and glycosyltransferases in the ER and Golgi.[14,18,19] These modifications generate complex, hybrid, or high-mannose glycan structures that influence protein folding, trafficking, and function.[20] Proper N-glycosylation ensures correct protein conformation and prevents premature degradation, while dysregulated N-glycosylation in cancer cells can lead to altered receptor activity, increased proliferation, and enhanced metastatic potential.[21] In cancer cells, alterations in NXS/T motifs, such as mutations, can disrupt normal glycosylation patterns. This may result in misfolded proteins, impaired cell signaling, and enhanced tumor progression.[22] Studies have shown that the strategic targeting of N-glycosylation sites in key oncogenic proteins, such as RTKs, could provide new therapeutic avenues by modulating receptor activity and downstream signaling pathways.[23] For example, in prostate cancer, N-glycosylation supports cross-talk between RTKs and the androgen receptor, thereby influencing cell viability and receptor activation.[24]

Recently, there has been a growing focus on the role of glycosylation in cancer progression, particularly in lung cancer. Notable examples include the use of fluorinated monosaccharides, which have demonstrated promising preclinical efficacy as potential cancer therapeutics.[25] In addition, targeting N-glycosylation has emerged as a strategy for cancer treatment, such as the application of 2-deoxyD-glucose to inhibit N-glycosylation in chimeric antigen receptor T cells. This approach reduces their binding affinity to immune-inhibitory molecules within the tumor microenvironment, thereby enhancing their cytotoxic activity against lung cancer cells expressing the cancer-testis antigen FMR1NB.[26] The use of 2-fluorofucose in cancer treatment trials has shown promising results in both human and animal studies, particularly in inhibiting protein and cellular fucosylation.[27] Glycosylation, particularly N-glycosylation, affects the function and stability of RTKs. These modifications play significant roles in various cellular processes, including signal transduction, protein folding, and cellular localization, which are crucial for both normal cellular functions and pathological conditions such as cancer. Inhibiting N-glycosylation has emerged as a potential therapeutic strategy to disrupt RTK signaling in cancer.[7] In lung cancer cells, alteration of glycosylation in RTKs such as EGFR, HER-2, c-MET, RET, ALK, and ROS1 can significantly impact their function and disease progression. This review summarizes the current advancements in our understanding of RTK glycosylation in cancer cells and its implications for treatment strategies that focus on lung cancer.

THE ROLE OF GLYCOSYLATION IN RTK SIGNALING EGFR

EGFR, encoded by ERBB1, is a critical RTK involved in the regulation of cell growth and survival. EGFR mutations are found in a significant proportion of NSCLC cases, with frequencies varying by region and population. EGFR mutations are more prevalent in females, Asian populations, and non-smokers, while smoking history inversely correlates with mutation frequency.[28-30] EGFR is structurally composed of three key domains: An extracellular domain (ECD) for ligand binding, a transmembrane domain for membrane anchoring, and an intracellular cytoplasmic domain containing catalytic tyrosine kinase activity.[31] In its inactive state, EGFR primarily exists as an auto-inhibited monomer. However, upon ligand binding, EGFR changes to a conformation that enables it to form either homodimers or heterodimers with HER-2 or other receptors. Receptor activation is possible upon the establishment of an asymmetric dimer configuration of the kinase domains, wherein one domain allosterically potentiates the activity of the other.[32] The EGFR ECD, which has a complex, heart-shaped structure, comprises 4 subdomains. Domain I (L1), with a leucine-rich repeat structure, is involved in ligand binding, as is Domain III (L2), which also has a leucine-rich repeat structure. Domain II (CR1) and Domain IV (CR2), which are cysteine-rich domains, contribute to the dimerization of the receptor. The entire ECD adopts a tethered conformation in the absence of a ligand, preventing spontaneous activation by keeping L1 and L2 in a compact state.[31,32] The intracellular domain of EGFR comprises a juxtamembrane region involved in tyrosine kinase domain regulation and function. It is a bilobal structure encompassing the ATP binding site (in the N-terminal lobe), the activation loop, and a substrate binding site (both in the C-terminal lobe). A C-terminal tail, rich in tyrosine residues, undergoes autophosphorylation and serves as a platform for interaction with downstream signaling proteins.[31,32] Oncogenic EGFR mutants induced by amplification of activating mutations exhibit functional characteristics similar to those of the ligand-activated wild-type receptor. Tumors with EGFR mutations often present with specific imaging features such as ground-glass opacity and histological patterns such as lepidic growth. These features can help predict genetic changes and guide treatment decisions.[33,34] The most prominent EGFR lesions involve the L858R point mutation in exon 21 and deletions within exon 19, often grouped as classical mutations that comprise 85-90% of EGFR kinase domain mutations.[35] In general, EGFR mutations and extensive rearrangements observed across solid cancer entities frequently cause dysregulation of receptor endocytosis, which, in turn, enhances the receptor’s signaling potential.[36]

EGFR is a heavily glycosylated protein and carbohydrate chains constitute approximately 25% of the 180 kDa mass of the receptor. This post-translational modification plays a fundamental role in various receptor processes, such as ligand-independent activation, growth factor binding affinity, and dimerization. Moreover, significant alterations in glycosylation are observed in various cancers, including NSCLC.[31] N-glycosylation is crucial for the proper folding, stability, and function of EGFR. Disruption of this process can lead to altered receptor activity and localization, which may enhance cancer cell proliferation and survival [Figure 2]. The absence of N-glycosylation can lead to increased dimerization of EGFR, even in the absence of ligands, which promotes spontaneous activation of the receptor. This can result in the activation of downstream signaling pathways that drive cancer cell proliferation and survival. EGFR-targeted therapies such as erlotinib can sensitize cancer cells, suggesting that targeting N-glycosylation could be a strategy to overcome drug resistance in EGFR-mutant cancers. Disruption of N-glycosylation in cancer cells can lead to uncontrolled receptor activation by promoting spontaneous oligomerization, ultimately contributing to increased proliferation, survival, and drug resistance.[37,38] It may also impair receptor maturation by altering subcellular localization.[39]

Figure 2: Epidermal growth factor receptor (EGFR) signaling upon disrupted N-glycosylation. EGFR dimerization and downstream signaling occur upon proper glycosylation of EGFR and ligand binding. This leads to EGFR pathway activation through Rat sarcoma (Ras), V-Raf Murine Sarcoma Viral Oncogene Homolog B (B-RAF), mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2), and extracellular signal-related kinase 1/2 (ERK1/2) activation resulting in cell proliferation, growth, and cell survival. Disruption of EGFR N-glycosylation enables ligand-independent dimerization and activation. P: Phospho; GTP: Guanosine triphosphate. Created in BioRender. Meder, L. (2025) https://BioRender.com/8rqpbva.

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The ECD of EGFR is heavily modified post-translationally by the addition of sugar, which influences receptor activity.[40] GlyGen, a web-based platform that integrates glyco-conjugate-related information from publicly available databases, shows 17 sites for N-glycosylation and 6 sites for O-linked glycans, but not all have been verified to be related to cancer.[41]

Glycosylation of the EGFR ECD occurs at Asn residues that fit the NXS/T consensus motif, including an atypical site at Asn32, which is located within a unique Asn-Asn-Cys sequence.[40,42,43] This atypical site has been shown to undergo both N-glycosylation and O-fucosylation. Experimental and computational analyses have demonstrated that glycosylation is essential for maintaining both the structural integrity and stability of the protein.[40] Through recent molecular dynamic simulations, the study of a complete monomeric glycosylated EGFR structure situated in a membrane bilayer has highlighted the critical role of N-glycosylation in enabling the EGFR ECD to adequately interact with the membrane, a prerequisite for maintaining its proper structural conformation.[42] Specific glycosylation sites, such as N361 in Domain III and N579 in Domain IV, are critical for EGFR dimerization. Mutations at N361A increase dimerization and alter ligand sensitivity, impacting cell viability and response to inhibitors.[44] Glycosylation at N579 affects the receptor’s conformation, influencing its dimerization state. The N579Q mutation, which prevents glycosylation, results in a higher fraction of receptors in a high-affinity state and alters phosphorylation patterns.[45] In total, 4 glycosylation sites are located in Domain III, where the monoclonal antibody cetuximab (an anti-EGFR targeted therapy) binds to its target epitope.[46] In addition, inhibition of N-glycosylation using OST inhibitors can overcome resistance to EGFR TKIs by disrupting EGFR signaling and reducing tumor cell viability. For instance, NGI-1, which is an OST inhibitor, was able to restore sensitivity to osimertinib, a first-generation TKI, by inducing G1 arrest and apoptosis in resistant NSCLC cells.[47] To date, O-glycosylation in EGFR signaling has not been studied in lung cancer so far. However, C1GALT1, which is responsible for elongation of GalNAc-type O-glycosylation, and N-acetylgalactosaminyltransferase 2 (GALNT2), which regulates the early steps of mucin O-glycosylation, have been shown to enhance oncogenic EGFR signaling in brain cancers and oral squamous cell carcinomas. Both glycosyltransferases trigger EGFR terminal O-glycosylation and promote EGFR phosphorylation and downstream signaling.[48,49]

In general, since EGFR-mutated cancer cells frequently develop resistance to EGFR blockade, attenuating the glycosylation of EGFR has emerged as a promising therapeutic approach for NSCLC. It may counteract dimerization and activation of overexpressed EGFR or can enable mAb binding. Therefore, exploring the inhibition of glycosylation as a complementary approach holds potential as a strategy to enhance therapeutic outcomes and possibly address drug resistance.

HER-2

HER-2 is an extensively studied member of the EGFR family of receptors in cancer research and is encoded by the ERBB2 gene. It is a transmembrane RTK that, when overexpressed or amplified, contributes to aggressive tumor behavior and poor prognosis.[50] HER2 mutations are identified in approximately 2% of NSCLC, with a notable prevalence in lung adenocarcinomas. These mutations are often found in specific subgroups such as females, non-smokers, and those with adenocarcinoma histology.[51]

HER-2 is a type I transmembrane growth factor RTK and exhibits significant structural similarity to other EGFR family receptors.[52] This class of receptors comprises an extracellular N-terminal domain that serves as the ligand-binding site, a transmembrane domain, and an intracellular region that includes a juxtamembrane domain, a kinase catalytic domain, and a carboxy-terminal domain. In contrast to other receptors, HER-2 lacks confirmed ligands for binding; its activation occurs through heterodimerization with other EGFR family receptors bound to their respective ligands or through homodimerization.

Amplification of ERBB2 and overexpression of HER-2 are well-established characteristics in breast, gastric, and lung cancers, serving as predictive markers for therapeutic approaches aimed at HER-2.[53] The most frequently observed and extensively studied alterations of HER-2/ERBB2 in tumor cells include protein overexpression as well as gene mutations and amplifications.[52-55]ERBB2 mutations are independent of amplification, indicating that they represent a separate biological entity and a potential therapeutic target.[55,56]ERBB2 overexpression or gene mutations result in an elevated presence of HER-2 on the cell surface and enhanced activation. This activation drives the uncontrolled proliferation of tumor cells.[50,53]

The percentage of NSCLC patients with ERBB2 mutations is approximately 1-4%, while ERBB2 gene amplification occurs in 2-5% of cases and protein overexpression in 2-30% of cases. This overexpression can be induced by cytotoxic drugs through downregulation of microRNAs miR-125a and miR-125b which normally suppress HER-2 expression. The majority of patients harboring ERBB2 mutations have a history of never smoking and present with adenocarcinoma histology.[57] The most frequent ERBB2 mutations are in-frame insertions within exon 20 (ex20ins), notably the A775_ G776insYVMA insertion/duplication. These mutations are typically found to be mutually exclusive with EGFR mutations and ALK rearrangements.[58]

The most common treatments for cancer patients with ERBB2 mutations and/or HER-2 overexpression involve a range of targeted therapies, including pan-HER-2 inhibitors (such as afatinib and neratinib) and targeted mAbs and antibody-drug conjugates such as trastuzumab and trastuzumab emtansine (T-DM1), respectively.[59] Trastuzumab, often used in combination with chemotherapy, has been utilized in treating HER-2-positive NSCLC. However, its efficacy as a monotherapy is limited, with a progression-free survival of about 5.1 months.[57,60] T-DM1 has shown superior activity against HER-2-expressing tumors compared to unconjugated trastuzumab, with improved efficacy and safety.[61]

There are studies suggesting that post-translational modifications of the HER-2 protein, rather than its overexpression, have a pivotal role in regulating endocytosis and determining the effectiveness of anti-HER-2 therapies in lung cancer cell lines and patient-derived xenograft models.[60] A recent study that utilized mass spectrometry to create a comprehensive glycosylation profile of HER-2 showed site-specific modifications and their impact on receptor stability and activity. HER-2 has 8 identified N-glycosylation sites in its ectodomain that can interfere with trastuzumab binding to its target epitope.[62] GlyGen references 6 sites for N-glycosylation: Asn124, Asn187, Asn259, Asn530, Asn571, and Asn629 and two sites for O-glycosylation: Thr127 and Ser998.[41] The effectiveness of trastuzumab in treating HER-2-positive gastric cancer is significantly challenged by molecular resistance, which is influenced by extensive N-glycosylation of the HER-2 receptor. Through mass spectrometry analysis, it was found that galactose α-2,6-sialyltransferase 1 (ST6Gal1) modifies specific N-glycosylation sites within the trastuzumab-binding domain of HER-2, affecting receptor stability and therapeutic response. Inhibiting ST6Gal1 expression altered glycosylation patterns, increased HER-2 stability, and enhanced sensitivity to trastuzumab by reducing HER-2 and EGFR activation.[63] Another way HER-2 targeting was used in the context of glycosylation, was coupling trastuzumab with a sialidase. The targeted sialidase activity toward breast cancer cells using antibody-enzyme conjugates led to desialylation of the cancer cell surface accompanied by increased NK cell killing by antibody-dependent, cell-mediated cytotoxicity.[64]

Taken together, these findings suggest a potential new area of investigation for HER-2 and other RTK modifications in tumor cells, focusing on the role of surface receptor glycosylation across various cancer types. Altered glycosylation patterns of HER-2, including modifications in the trastuzumab-binding domain, can significantly influence receptor stability and activity as well as impact the efficacy of targeted therapies. In addition, directing de-glycosylating enzymes to RTKs might indicate a novel approach to improve immunotherapeutic approaches in cancer treatment in the future, including lung cancer treatment.

c-MET

c-MET mutations occur in approximately 3-5% of NSCLC cases, with a higher prevalence in adenocarcinoma and a notable enrichment in the sarcomatoid subtype. In addition, de novo c-MET amplifications are observed in 1-5% of NSCLC, primarily affecting adenocarcinoma.[65] c-MET is an RTK, and its active form is a disulfide-linked heterodimer, which includes an extracellular α-subunit and a single-pass transmembrane β-subunit. The extracellular portion of c-MET features a large N-terminal semaphorin (SEMA) domain, an integrin PSI domain, along with transcription factor IPT domains. The intracellular portion of c-MET consists of three main segments: a juxtamembrane region, a tyrosine kinase domain, and a C-terminal docking site.[66] c-MET activation occurs when it forms a homodimer in response to binding with a ligand. This binding involves high-affinity sites located within the IPT3 and IPT4 domains, as well as low-affinity sites within the SEMA domain.[67] Hepatocyte growth factor (HGF) is the only known true endogenous ligand for c-MET.[68] Human c-MET contains several reported glycosylation sites: 10 N-linked and three O-linked sites, according to GlyGen.[41]

Some findings suggest that the N-glycans of the SEMA domain positively regulate HGF signaling, whereas the N-glycans of the other regions negatively regulate signaling.[69] Specific N-glycans on c-MET regulate its proteolytic processing and HGF-induced signaling in a site-specific manner. Deletion of these N-glycans affects c-MET’s status and function. c-MET is initially synthesized as a partially glycosylated single-chain precursor (pro-MET) in the ER. Pro-MET undergoes disulfide bond formation, post-translational glycosylation, and endoproteolytic cleavage to produce the mature heterodimeric form.[70] During receptor homodimerization and autophosphorylation, the activation of several downstream signaling pathways occurs, including the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and RAS/extracellular signal-regulated kinase (RAS/ERK) pathways. These signaling pathways drive various cellular processes, such as cell proliferation, migration, differentiation, and morphogenesis.[69]

Dysregulation of c-MET, resulting in aberrant signaling, can occur through various mechanisms such as gene amplification, overexpression, activating mutations, increased autocrine or paracrine ligand stimulation, HGF overexpression, formation of abnormal autocrine signaling, and interaction with other active cell-surface receptors.[71] Elevated c-MET expression has been observed in multiple cancers, including lung, breast, ovarian, kidney, colon, thyroid, liver, and gastric carcinomas.[68] Approximately 3-5% of NSCLC patients have c-MET dysregulation, including splice site mutations in METex14 that have been reported in about 4% of NSCLC patients.[68,71,72] Recent advancements have led to the development and approval of several c-MET TKIs, such as capmatinib and tepotinib.[73-75] Drug resistance is an unavoidable challenge in targeted therapies. The presence of MET amplification can lead to short-lived and heterogeneous responses.[76]MET amplification is a well-established mechanism of acquired resistance to EGFR TKIs in NSCLC patients. It often occurs alongside secondary EGFR mutations, contributing to the complexity of resistance.[77]

Since both transcriptional and post-translational mechanisms control the expression and activity of c-MET, NSCLC treatment may benefit from a combination of c-MET TKIs with drugs that influence c-MET maturation in cancer cells. C1GALT1 controls the formation of mucintype O-glycans, including O-glycosylation of c-MET, thereby enhancing HGF-induced dimerization and activation.[78] GALNT2, an enzyme involved in the first step of mucin-type O-glycosylation, inhibits malignant characteristics in gastric adenocarcinoma by modulating the activity of the c-MET receptor. Knocking down GALNT2 significantly increased c-MET phosphorylation, especially in response to HGF stimulation, and reduced lectin binding to c-MET O-glycans, indicating that GALNT2 plays a role in modifying c-MET O-glycans in gastric cancer cells.[79]

Taken together, specific N-glycans in the SEMA domain enhance HGF signaling, while those outside of this domain suppress it. O-glycosylation, particularly mediated by C1GALT1 and GALNT2, influences c-MET dimerization, activation, and downstream signaling pathways. These modifications suggest that targeting glycosylation could become a novel therapeutic option for NSCLC. Combining c-MET inhibitors with drugs targeting c-MET maturation, such as glycosylation modulators, could enhance treatment efficacy and might improve outcomes for NSCLC patients with c-MET-driven tumors.

RET

RET rearrangements occur in approximately 1.2-2.2% of NSCLC cases, with a slightly higher prevalence in adenocarcinomas.[80,81] RET is distinguished from other RTKs by its extracellular region. This domain is characterized by 4 cadherin-like structures and a segment of 120 amino acids containing 16 cysteine residues.[82] It is activated by glial cell line-derived neurotrophic factor (GDNF) ligands binding to GDNF family receptor alphas, leading to RET dimerization and activation. This activation triggers downstream signaling pathways such as PI3K/AKT, mitogen-activated protein kinase, and phospholipase C gamma (PLCγ), promoting cell proliferation, growth, and survival.[82,83] Overexpression and rearrangements of RET have been identified as potential oncogenic drivers, leading to increased interest in targeted therapies. RET rearrangement is observed in approximately 1-2% of lung adenocarcinomas, with reports indicating that activating EGFR mutations can also co-exist.[84,85] RET is also known for its fusions, such as kinesin family member 5B (KIF5B)-RET, which are found in about 1.7% of lung adenocarcinomas and often associated with younger, non-smoking patients and poorly differentiated tumors.[86] Targeted therapies, such as RET TKIs selpercatinib and pralsetinib, show antitumor activity in lung tumors with high RET expression. Vandetanib, a multikinase inhibitor, may overcome resistance to EGFR-TKIs in RET-overexpressing lung adenocarcinomas, suggesting its potential use in combination therapies.[87]

At present, there is a lack of studies specifically addressing the glycosylation of RET in cancer and the potential functional consequences. However, GlyGen identifies 4 reported N-glycosylation sites of RET: Asn343, Asn554, Asn763, and Asn975.[41]

ALK and ROS1

ALK rearrangements occur in approximately 3.8-6.3% of NSCLC cases, with a higher prevalence in adenocarcinomas. ALK-positive NSCLC is more common in younger patients, women, and never-smokers. The mean age difference between ALK-positive and -negative patients is significant, with ALK-positive patients being younger by about 7 years.[88] ALK is a receptor transmembrane protein tyrosine kinase and is involved in various signaling pathways that regulate cell growth, survival, and differentiation by its activation through binding of the extracellular ligands ALKAL1 and ALKAL2.[89,90] Like many RTKs, ALK is structured with three distinct regions: an extra-cellular domain that binds to ligands, a single-transmembrane segment that anchors the protein, and an intracellular cytoplasmic tyrosine kinase domain critical in the development of diseases.[91] ROS1 is also an RTK that has been implicated in various cancers, but its exact function in healthy cells remains unknown. ROS1 and ALK share significant structural similarities, particularly in their kinase domains, which have 49% amino acid sequence homology. This substantial overlap contributes to their functional similarities.[92,93]

ALK fusions, generated by chromosomal rearrangements, can signal continuously without the normal regulatory controls. The first alteration of the ALK gene identified in human cancers was the nucleophosmin-1 (NPM1)-ALK fusion gene.[89] In t