3.1 Ligand-induced dimerization of RTKs

The preformed dimer of the receptor can exist in either an “inactive” or “active” state, as the receptor maintains a dynamic balance between the dimer and the monomer. It is likely that the “inactive” and “active” dimers are in a state of dynamic equilibrium. When a ligand binds to the receptor, it can alter this equilibrium and induce dimerization.^52-54 There are generally four modes of RTK dimerization that lead to the activation of tyrosine kinase structural domains. These modes can be categorized into two mechanical extremes and two intermediate cases. In one extreme, exemplified by TrkA, receptor dimerization is solely mediated by the ligand and does not involve any direct contact between the two receptors.⁵⁵ On the other extreme, dimerization is entirely mediated by the receptors themselves, without any physical interaction between the two activating ligands, as seen in ErbB family members.⁵⁶ An intermediate situation occurs when a ligand homodimer binds to two receptor molecules before interacting with them through the dimer interface, as observed in the case of KIT.⁵⁷ Another intermediate circumstance arises when an auxiliary molecule not only binds bivalent ligands and makes direct receptor-receptor interactions but also participates in receptor dimerization. For example, the FGFR family utilizes heparin or heparan sulfate as an auxiliary molecule in this mode.^{58, 59}

3.2 Activation of intracellular TKDs

Prior to activation, TKD is inhibited in a cis conformation through specific intramolecular interactions.^{60, 61} This cis autoinhibition can be relieved by ligand-induced dimerization. Each TKD's receptor has distinct intramolecular interactions that contribute to cis autoinhibition. The pivotal step in RTK activation occurs when the cis autoinhibition is released upon ligand-induced receptor dimerization.

The self-inhibition of RTKs can be observed through the activation loop structure of the insulin receptor TKD.⁶² In this study, a novel finding is presented, demonstrating that the activation loop structure of the insulin receptor TKD is connected to a crucial tyrosine (Y1162) in the kinase. This connection serves to stabilize the activation loop structure, blocking the active site and impeding access to ATP and protein substrates. Consequently, the insulin receptor TKD is self-inhibited in cis through its own activation loop. However, when the receptor is engaged, trans-phosphorylation disrupts the cis-autoinhibitory connections, leading to the relaxation of the insulin receptor's TKD into an activated state. This process is achieved through autophosphorylation, which effectively “releases” the cis-autoinhibition. It is worth noting that activation loop autoinhibition also impacts other receptors, such as FGFR and IGF-1R, in addition to the insulin receptor.^{63, 64}

Juxtamembrane domain autoinhibition is an additional regulatory mechanism that complements the activation loop. The juxtamembrane domain sequences play a crucial role in interacting with various sections of the TKD, thereby stabilizing an autoinhibited conformation. This mechanism is responsible for controlling KIT and Eph receptors.^{61, 65} The self-inhibitory connections can be disrupted by phosphorylating specific tyrosine residues in the juxtamembrane domain, leading to the adoption of an active conformation by TKD.⁶²

C-terminal tail inhibition refers to the process where the C-terminal tail of receptors such as TEK, MET, and RON (MST1R) interacts with the active site of TKD, preventing the entry of substrates.⁶⁰ In the case of Tie2, the nucleotide-binding loop adopts an inactive conformation, and the C-terminal tail region containing the tyrosine autophosphorylation site hinders substrate access to the active site. This interaction stabilizes the inactive conformation and effectively inhibits the kinase activity. However, when ligand-induced dimerization occurs, the kinase adopts an active configuration by undergoing trans-phosphorylation of important tyrosine residues. This process disrupts the self-inhibitory connections, leading to the activation of the kinase.

3.3 Mechanism of activation of downstream signaling

The activation of RTK and subsequent autophosphorylation of this protein leads to the completion of various downstream signaling proteins. The autophosphorylation event in “phase I” primarily serves to enhance the catalytic activity of the kinase after the receptor binds to its activating ligand. The “phase II” autophosphorylation events require prior activation of the kinase in “phase I” and the formation of a binding site based on phosphotyrosine. This binding site recruits cytoplasmic signaling molecules that contain Src homology-2 (SH2) and phosphotyrosine-binding (PTB) domains.^{50, 66} Upon recognition of both types of proteins, RTK functions as a kinase, transferring its own phosphate to both proteins and leading to subsequent intracellular effects. The activated RTK has the ability to attract and regulate multiple signaling pathways due to the presence of numerous phosphotyrosines and the involvement of various docking proteins. For instance, in the case of MET, these downstream signaling pathways include important ones such as RAS/MAPK, PI3K/AKT, and STAT3 (Figure 1).⁶⁷ Consequently, RTK activates transcriptional pathways that play a role in controlling various cellular activities and acts as a central hub for transmitting complex information about cell growth and migration from the extracellular environment to the nucleus.

4.1 Gain-of-function mutations

Acquired functional mutations have altered the downstream signaling of the RTK gene, resulting in its suppression no longer being the norm. Mutations in proto-oncogenes can provide cells with a selective growth advantage. These driver genes not only offer the potential for targeted treatment of malignancies but also contribute to our understanding of the mechanisms involved in carcinogenesis and development. Somatic mutations in genes encoding RTKs often occur in evolutionarily conserved residues, such as the kinase activation loop and the DFG motif surrounding the nucleotide binding pocket. These conserved residues (D, F, and G) play crucial roles in ATP binding and catalytic activity.^{70, 71}

Notable examples of the mutation spectrum in RTKs can be observed in MET somatic cell mutations. Variants within the MET exon 14 region consist of various nonhomologous mutations that can lead to the activation and perpetuation of the MET pathway. Among these mutations, base substitution and insertion loss are equally prevalent and significant contributors to the development of cancer.^{72, 73} Upon activation of MET protein, its Y1003 site is typically phosphorylated.⁷⁴ This phosphorylation event enables MET to bind with c-Cbl E3 ligase, resulting in the ubiquitination modification of MET protein. The modified protein is then degraded, thus forming a self-regulated negative feedback loop.⁷⁵ The skipping of MET exon 14 results in the loss of ubiquitination of MET proteins due to mutations in its receptor binding site. This deletion causes the removal of the E3 ubiquitin ligase c-Cbl, which in turn increases the instability of MET proteins. As a result, downstream signaling pathways are activated, leading to tumorigenesis. Although MET exon 14 alterations were originally identified in small cell lung cancers (SCLCs),⁷⁶ subsequent studies have found that these genetic variants are more common in NSCLC, accounting for approximately 3−4% of cases.^{72, 73, 77, 78}

Mutations can also occur in the extracellular structural domain, transmembrane structural domain, and near-membrane structural domain of RTK. These mutations are linked to an elevated expression of RTKs, resulting in their phosphorylation even in the absence of ligand stimulation.⁷⁹ In 1997, mutations in the structural domain of MET kinase were first reported in hereditary papillary renal carcinoma.⁸⁰ These mutations, which include V1092I, H1094R/Y, M1131T, V1188L, V1220I, M1250T, and D1228H/N/V, have been shown to increase kinase activity and may modify phenotype or form tumor foci.⁸¹ These mutations have also been found to induce tumorigenesis in mouse models.^{81, 82} Twenty years later, somatic MET kinase mutations, including V1092I, H1094L/R/Y, N1100Y, H1106D, M1131T, V1188L, L1195V, V1220I, D1228H/N/V, Y1230A/C/D/H, Y1235D, and M1250I/T, in the structural domain were found in sporadic papillary RCC (PRCC).^{80, 83, 84} These mutations, which were accompanied by MET amplification, showed lower activity. Similarly, mutations in the structural domain of MET kinase have also been observed in other tumors like hepatocellular carcinoma (HCC) and head and neck tumors.^{82, 85, 86}

4.2 Overexpression and genomic amplification

RTKs are frequently found to be overexpressed in various types of human cancers. This overexpression leads to an increased concentration of the receptor in the affected area, which in turn results in the activation of RTKs signaling pathways. These pathways play a crucial role in counteracting antagonistic regulation.⁸⁷ There are two ways in which an increase in MET copy number can occur: through multimerization or amplification. Polymorphism arises when the replication of chromosome 7, where MET is located, is not properly carried out.^{88, 89} Polyploidy cannot be considered as a driver gene. Instead, MET amplification is defined as a local acquisition with local gene duplication. This type of amplification can effectively enhance the MET copy number without replicating chromosome 7.⁹⁰ Therefore, compared with the broad increase on chromosome 7, the focused MET amplification more accurately represents the real oncogenic driving state.⁹¹ Preclinical studies have shown that MET amplification can lead to upregulated MET expression, ligand-independent receptor activation, and downstream signaling.^{92, 93}

Gene amplification is the main mechanism that leads to the overexpression of RTKs. However, RTKs can also be overexpressed through transcriptional/translational enhancement,⁹⁴ oncogenic viruses,⁹⁵ derailments of normal regulatory mechanisms such as loss of phosphatases⁹⁶  or other negative regulatory factors.^{97, 98}

4.3 Chromosomal rearrangements

New tyrosine kinase fusion oncoproteins are formed due to various chromosomal rearrangements.^{78, 99, 100} As a result, small molecule inhibitors are often developed to target these abnormal fusion proteins for therapeutic purposes. The first tyrosine kinase fusion to be identified was BCR–ABL. This fusion resulted from the translocation t (9,22), also known as the “Philadelphia chromosome”. It involved the joining of the ABL1 tyrosine kinase genes on chromosome 9 with the BCR gene on chromosome 22, creating the BCR–ABL fusion oncoprotein.¹⁰¹ Patients with chronic myelogano leukemia (CML) and certain patients with acute lymphoblastic leukemia are known to have BCR–ABL fusion.^{102, 103} Subsequently, the introduction of imatinib, a medication that targets the ABL kinase, revolutionized the treatment of CML patients and marked the beginning of targeted therapies.^{104, 105}

While BCR–ABL is primarily observed in leukemia, numerous tyrosine kinase fusions have been identified in various types of tumors, encompassing both liquid and solid malignancies. For example, MET fusions have been identified in various types of cancer such as osteosarcoma,¹⁰⁶ gastric cancer,¹⁰⁷ thyroid cancer,¹⁰⁸ lung cancer,¹⁰⁹ glioma,¹¹⁰ and sarcoma.¹¹¹ In 1984, the MET gene was first identified in a chemically induced human osteoma cell line as part of an oncogenic fusion with the translocated promoter region (TPR) gene.¹¹² Apart from TPR–MET, which was initially discovered in osteosarcoma.¹¹² numerous other MET fusions have also been documented.^{99, 113-121} MET fusions can be achieved by either interchromosomal fusions, such as KIF5B–MET, or intrachromosomal fusions, such as PTPRZ–MET. These fusions typically consist of the kinase region, which is encoded by exon 15 of the MET gene, and the dimerized regions of various upstream partner genes.^{99, 113, 115, 116, 120, 122-124} This leads to an activation of MET substitution that is independent of ligands. The PTPRZ promoter is commonly associated with the complete MET gene in the PTPRZ–MET fusion, resulting in elevated expression of MET and activation of its downstream signaling pathways.^{122, 125}

4.4 Autocrine activation

Growth factors and cytokines serve as messengers in cell-cell communication, transmitting signals from the secreting cell to a distant target cell. When the target cell secretes the cell itself, it is referred to as “autocrine.” Constitutive autocrine activation can lead to clonal growth and tumor development. In various malignancies, autocrine activation of several RTKs such as TGFα-EGFR,¹²⁶ HGF-MET,^{127, 128} and SCF-KIT^129-131 is well documented. Taking the HGF-MET autocrine loop as an example, sustained activation of multiple MET proteins occurs in a ligand-dependent manner.¹³² HGF-producing and expressing cells engage in crosstalk within the TME, including between the stromal and epithelial components, as well as among stromal cells like tumor-associated macrophages and neutrophils.¹³³ It is noteworthy that many stimuli that trigger MET transcription in cancer cells also lead to HGF upregulation in the tumor stroma, creating a feedforward stimulatory circuit that enhances MET activation.^{127, 128} Autocrine mechanisms can be a promising target for cancer therapy.¹²⁶ One example is the case of EGFR-mutated lung cancer cells, which show reduced responsiveness to EGFR-tyrosine kinase inhibitor (TKI) due to the presence of a ligand/receptor autocrine loop.¹³⁴

4.5 Alterations in the TME

The TME refers to the internal environment in which tumor cells develop and exist. It plays a crucial role in the development of cancer.¹³⁵ One of the important cellular components of the TME is macrophages. AXL, a protein, contributes to immune suppression and the development of a preneoplastic phenotype, which is highly expressed in macrophages associated with tumors.¹³⁶ The protein HGF is primarily secreted by cancer-associated fibroblasts (CAFs) and has a paracrine effect on nearby tumor cells.^{137, 138} When HGF binds to MET, it triggers the auto-phosphorylation of tyrosine residues (Tyr1234, Tyr1235) in the cytoplasm. This, in turn, activates the intracytoplasmic protein tyrosine kinase (PTK) structural domain of c-Met, leading to the phosphorylation of c-Met C-terminal tyrosine (Tyr1349, Tyr1356). During cellular processes, substrate proteins undergo phosphorylation reactions leading to their polymerization and abnormal activation. This results in changes in cell proliferation, survival, apoptosis, invasion, migration, and blood vessel formation. Reactive ligands with high bioavailability bind to their highly expressed active receptors, becoming a common mechanism for antitumor drug targeting action. This adaptive mechanism is particularly important in tumor tissues characterized by hypoxia and inflammation. Many other RTKs have also been shown to play important roles in the TME, including the Eph receptor family, VEGFR and PDGFR.^139-141

4.6 RTKs as stem cell markers

It is well established that a subset of cancer cells known as cancer stem cells (CSCs) contributes to tumor heterogeneity, metastasis, and resistance to treatment.¹⁴² RTKs play a crucial role in maintaining the characteristics of CSCs, such as self-renewal capacity, viability, invasiveness, and tumorigenicity. Several RTKs have been identified to be involved in CSC maintenance, including Eph receptor,¹⁴³ MET,¹⁴⁴ EGFR,¹⁴⁵ and others. Taking MET as an example, it is primarily expressed in stem and progenitor cells in normal tissues, while its expression is absent in cells that possess the ability to differentiate. As a result, it is believed that MET can be used to identify the functional status of CSCs since it is widely expressed in certain cancers, which could serve as a marker for the proliferation of cells with stem or progenitor cell characteristics.¹⁴⁶

MET has the ability to promote stem cell-like functions that are crucial for tumor initiation, multiplication, regeneration, and dissemination. This activity occurs regardless of whether MET is oncogenically activated or has prosurvival activity. In pancreatic cancer, MET serves as both a marker and therapeutic target for CSCs,¹⁴⁷ Additionally, the interaction between HGF/MET and tumor-stromal cells helps to maintain the preferred glucose metabolism of pancreatic tumor stem cells.¹⁴⁸

The interaction between HGF and MET can cause abnormal expression of the wnt/β-catenin pathway, leading to metastasis in colon cancer.¹⁴⁹ Meanwhile, in HCC, the MET/FRA1/HEY1 cascade reaction, which is activated in CAFs, plays a vital role in regulating self-renewal in CSCs.¹⁵⁰ By encouraging cancer cells to migrate through the epithelial-mesenchymal transition (EMT) process, activation of the MET pathway plays a critical role in boosting the preservation of CSCs within tumors and enhancing metastasis.^151-153

5.1 Breast cancer

Breast cancer is the leading cause of morbidity and mortality among women worldwide. The development of breast cancer is attributed to the deregulation of multiple signaling pathways in the breast epithelial cells. Activation of various signaling cascades by growth factors and chemokines occurs in the TME, ultimately promoting tumor growth. VEGFRs, EGFRs, FGFRs, and PDGFRs are present in different malignancies, including breast cancer. Elevated levels of RTKs have been associated with increased aggressiveness of breast cancer and lower overall and disease-free survival (DFS).¹⁵⁴

The significance of angiogenesis in the development of breast tumors has been extensively studied. VEGF, a potent proangiogenic factor, stimulates both lymphangiogenesis and tumor angiogenesis by binding to three different types of VEGFRs.^155-157 The expression of VEGFR1 was found to be significantly higher in breast tumor tissues, regardless of lymph node metastasis, compared with benign tumors or surrounding healthy tissues.¹⁵⁸ VEGF induces the production of VEGFR2, which then activates the JAK2/STAT3 signaling pathway, leading to the upregulation of Myc and Sox2 expression. In triple-negative breast cancer (TNBC), the autocrine loop formed by the VEGF/VEGFR2 axis involving STAT3, Myc, and Sox2 contributes to the enhancement of the tumor stem cell-like phenotype.¹⁵⁹ Neolymphatic vascular development plays a critical role in the spread of cancer cells and the formation of distant metastases. Therefore, targeting the growth of new lymphatic vessels has emerged as a potential therapeutic strategy for breast cancer. However, the progress in developing antilymphangiogenic treatments for different types of cancer has been impeded by the lack of suitable markers to accurately assess lymphatic vessels and lymphatic metastases.¹⁶⁰ VEGFR3 is an RTK that is expressed on lymphatic endothelial cells and plays a crucial role in the process of lymphangiogenesis.⁵⁵ The VEGF-C/VEGFR3 axis promotes lymphangiogenesis and the expression of VEGFR3. In the context of postnatal breast cancer, COX-2 facilitates the metastasis of lymph nodes by promoting lymphangiogenesis and upregulating VEGFR3 expression.^{161, 162} Galectin-8-mediated crosstalk between the VEGF-C, podoplanin, and integrin pathways plays a crucial role in lymphangiogenesis, and the presence of VEGFR3 is vital for this process.¹⁶³ Targeting lymphangiogenesis by using anti-VEGFR3 could potentially extend patient survival and decrease the dissemination of malignant cells.

Breast cancers with higher levels of EGFR overexpression have been associated with increased aggressiveness and poorer clinical outcomes. EGFR activates numerous downstream signaling molecules that promote cell proliferation, survival, and tumor progression. In a study conducted on 220 breast cancer patients, immunohistochemistry was employed to examine the expression of EGFR1, HER2, EGFR3, and EGFR4. The study revealed that EGFR1 was overexpressed in 16.4% of the tissues, HER2 in 22.8% of the tissues, EGFR3 in 17.5% of the tissues, and EGFR4 in 11.9% of the tissues.¹⁶⁴ Upon binding to ligands, EGFR activates several downstream signaling molecules, including RAS, PI3K, phospholipase C-γ (PLC-γ), and JAK. This activation leads to cell survival, cell growth, and tumor progression.^{145, 165, 166} Inflammatory breast cancer (IBC) is characterized by NF-κB activation, which causes endoplasmic reticulum (ER) downregulation, overexpression of EGFR and ErbB2, and hyperactivation of MAPK.¹⁶⁷ Additionally, Nodal signaling, regulated by the EGFR/cyclooxygenase-2 (COX-2) axis, promotes the CSC phenotype and enhances the invasive capacity of IBC cells by inducing EMT.¹⁶⁸ Animal models have shown that TAMs activation in cancer cells leads to STAT3-mediated Sox2 expression, resulting in an increase in the number of tumor stem cells and metastasis in a mouse breast cancer model.¹⁶⁹

Upon ligand stimulation, members of the FGFR family can activate pathways such as RAS/MAPK and PI3K/AKT, which play crucial roles in cell survival, proliferation, apoptosis, and migration. Notably, FGFR1 gene amplification has been observed in metastatic lobular breast cancer, ER⁺ breast cancer, and HER2⁻ breast cancer.^{170, 171} Furthermore, the expression of FGFR2 and FGFR3 is associated with the progression of ER+ breast cancer.¹⁷² FGFR4 and ErbB2 collaboratively regulate cyclin D1 expression, thereby promoting cell proliferation in breast cancer.¹⁷³ These findings highlight the interconnectedness of FGFRs with other RTKs and their implications in resistance mechanisms. Consequently, targeting FGFRs holds promise as a therapeutic approach for breast cancer.

PDGFRs, which include both PDGFR-α and PDGFR-β family members, are found to be highly expressed in breast tumors and mesenchymal cells. Numerous studies have indicated that PDGFR expression is correlated with a poor prognosis in breast cancer patients, suggesting its prognostic and predictive potential.^174-176 Moreover, PDGFRs have also been observed in reactive connective tissue mesenchyme, implying their potential role in tumor–mesenchymal interactions alongside their direct influence on cancer cells.^175-177 Therefore, targeting PDGF/PDGFR in the TME holds promise as a therapeutic strategy for the treatment of TNBC.

5.2 Prostate cancer

FGF plays a complex role in the physiopathology of the prostate, from fundamental roles throughout embryonic development to regulation of tumor transformation. Maintaining a healthy FGF/FGFR signaling axis in the adult prostate is crucial for maintaining organ homeostasis and function. Disruption of this signaling axis can lead to prostatic hyperplasia and may promote cancer development and metastasis. Studies on FGF/FGFR can help overcome the challenges of treating prostate tumors.

While FGFR-activating mutations are involved in various human tumors, prostate cancer commonly exhibits ectopic/altered levels of FGFR expression. Malignant prostate tumors frequently show ectopic expression of FGFR1, which triggers an autocrine loop due to aberrantly expressed FGFs.^178-180 This autocrine loop promotes cancer cell autopoiesis, stimulates cell proliferation and migration, and prevents cancer cell death.¹⁸¹ Furthermore, ectopic FGFR1 has the ability to reorganize cellular energy metabolism in prostate cancer cells¹⁸² and stimulate inflammatory responses by activating NF-κB signaling.¹⁸³ As prostate cancer progresses and differentiation decreases, FGFR1 expression increases.¹⁸⁴ Various in vivo studies have shown that FGFR2 expression either suppresses prostate cancer growth and progression or has no effect, in contrast to the significant role played by FGFR1 in this process. In fact, the absence of FGFR2 expression affects the mesenchymal-epithelial signaling axis and is associated with prostate cancer development.¹⁷⁸ The restoration of FGFR2 in human prostate cancer cells enhances their sensitivity to chemotherapeutic agents.¹⁸⁵ Therefore, in mouse models of prostate cancer cells, chemically induced FGFR1 dimerization/activation leads to rapid tumor growth, while inducible FGFR2 expression slows down tumor growth. Some low-grade prostate tumors exhibit somatic mutations in the FGFR3 gene, suggesting its involvement in FGF signaling in prostate cancer.¹⁸⁶ FGFR4 is expressed in tubular epithelial cells of penile intraepithelial neoplasia and prostate cancer tissues.¹⁸⁷

Bone metastases in patients with advanced prostate cancer are a life-threatening condition. The development of bone metastases depends on the complex interactions among prostate cancer cells, osteoblasts, osteoclasts, and the bone matrix.¹⁸⁸ Prostate cancer cells release growth factors and cytokines that stimulate bone cells.^{189, 190} These factors prompt osteoblasts to multiply and deposit new bone tissue. Consequently, osteoblasts and bone-derived substances are released, which further stimulate the proliferation of cancer cells and activate osteoblasts. Throughout the osteogenic spectrum, FGF plays a critical role in regulating bone formation.^{191, 192} The interaction between FGFRs and FGF2 in osteoblasts triggers the activation of the p42/44 MAPK and PKC signaling pathways. PKC activation leads to increased expression of RUNX2, a transcription factor essential for osteoblast development.

The molecular mechanisms underlying FGF/FGFR-mediated modulation of angiogenesis in prostate cancer were investigated through various preclinical studies conducted in different experimental settings. One such study involved in vitro and in vivo tests, which demonstrated that downregulating FGF2 in TRAMP-C2 cells by overexpressing the tumor suppressor SEF-b resulted in decreased angiogenesis.¹⁹³ Additionally, activating FGFR1 conditionally in mouse prostate epithelium induced angiogenesis by upregulating HIF-1α, VEGF, and angiopoietin-2.¹⁹⁴ These reports provide a theoretical basis for utilizing the suppression of the FGF/FGFR system as a combined antitumor/antiangiogenic therapy, with potential clinical implications for prostate cancer treatment. Overall, these findings suggest that the FGF/FGFR system plays a significant role in neointima formation in prostate cancer.¹⁹⁵

Lymphangiogenesis plays a crucial role in the metastasis of tumors to both regional lymph nodes and distant organs.^{196, 197} Its association with tumor progression and unfavorable prognosis in prostate cancer has been well established.^198-200 Studies have revealed that FGF2, derived from prostate tumor cells, can potentially stimulate lymphocytes by activating the FGFR1/AKT/mTOR/p70S6 kinase pathway.²⁰¹

5.3 Lung cancer

Following the 2015 World Health Organization classification, lung cancer is now commonly referred to as NSCLC, while SCLC is included in the new category of neuroendocrine tumors.²⁰² NSCLC can be further divided into three distinct subgroups: squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. The majority of patients with NSCLC are diagnosed at an advanced stage, and their median survival time after diagnosis is typically less than one year.²⁰³

The identification of the echinoderm microtubule-associated protein-like protein 4 (EML4)-ALK fusion gene in a group of NSCLC patients was first reported in 2007. Oncogenic ALK rearrangements result in ALK overexpression and constant activation, independent of ligands, by connecting the intact kinase structural domain of ALK to the Amino-(N-)terminal region of its partner. While other fusion partners of ALK have been found, EML4–ALK is the most prevalent type.²⁰⁴ ALK rearrangements occur in 3−7% of NSCLC, primarily in the lung adenocarcinoma (LUAD) subtype.²⁰⁵ Although ALK-positive NSCLC patients represent a small percentage of all NSCLC cases, they contribute significantly to the overall number of lung cancer cases worldwide. Studies have demonstrated the oncogenic potential of EML4–ALK in the development of lung cancer in mice. By overexpressing EML4–ALK in lung type II alveolar cells, researchers observed rapid tumor growth with LUAD characteristics.^{206, 207} Another study induced EML4–ALK rearrangements in vivo using CRISPR/Cas9 gene editing, which also led to the formation of lung tumors.²⁰⁸ These animal models are valuable as they exhibit sensitivity to ALK inhibition, allowing for the exploration of the mechanisms underlying EML4–ALK-induced lung cancer and the evaluation of ALK-targeted therapy's effectiveness.

The dysregulation of the kinase activity of the EGFR protein can occur due to various oncogenic processes, including EGFR gene mutations, increased gene copy number, and overexpression of the EGFR protein.²⁰⁹ The EGFR family of receptors and ligands plays a crucial role in the complex interactions between tumor cells and the TME. Additionally, EGFR may interact with the integrin pathway and activate matrix metalloproteinases, which can modify cell adhesion, stimulate cell motility and invasion, and facilitate metastasis.²¹⁰ Among the originally reported tumors with EGFR mutations, three were classified as fine bronchioloalveolar carcinomas, suggesting a higher prevalence of EGFR mutations in adenocarcinomas of the lung with features of fine bronchioloalveolar carcinoma.²¹¹ While some studies have yielded similar findings, others have not definitively established a link between EGFR mutations and specific subtypes of adenocarcinoma.^212-215 Furthermore, investigations have shown the presence of EGFR mutations in healthy fine bronchial epithelium alongside mutation-positive adenocarcinomas, indicating that, in certain cases, EGFR mutations may occur early in the development of LUAD.²¹⁶

MET, a receptor protein mainly found in epithelial cells, plays a crucial role in embryogenesis, tumor growth, and metastasis.²¹⁷ The MET proto-oncogenes encode these receptor proteins, which regulate genetic programs associated with cell proliferation and invasion of the extracellular matrix. In NSCLC, primary MET gene amplification has been observed in 1−5% of cases. However, it is more significant as it is also a mechanism of acquired resistance to EGFR-TKIs, observed in 5−20% of cases.¹³³ Another way MET activation occurs in NSCLC is through point mutations, particularly the elimination of exon 14, which is observed in 2−4% of NSCLCs and is more common in lung sarcomatoid carcinomas.^{73, 218} These mutations have also been linked to other malignancies such as gastric cancer (7%) or CRC (0–9%).⁷² It is worth noting that MET alteration is a recurring and actionable mechanism of resistance in ALK-positive lung cancer.²¹⁹ Also, KRAS acquired bypass resistance mechanisms include MET amplification.²²⁰

5.4 Colorectal cancer

Bowel, colon, and rectal cancers are commonly classified as CRC due to their shared characteristics. Metastasis is the primary factor contributing to cancer-related deaths in patients with CRC. The majority of colon cancer cases originate from small, benign adenomatous polyps that eventually become malignant. While conventional chemotherapy has significant benefits in cancer treatment, the development of secondary resistance and nonspecific toxicity to rapidly dividing cells pose major challenges in achieving optimal outcomes. However, the field of molecular oncology has made remarkable advancements, enabling the development of highly selective medications that induce cancer cell death by targeting specific genes or proteins involved in cell proliferation or antiapoptosis.²²¹ RTKs play a crucial role in the development of CRC (Figure 3).

In CRC cell lines, the PDGFR gene is frequently found to be overexpressed or mutated.²²² The overexpression of PDGFR has been identified as a useful biomarker for detecting and managing CRC. It has been linked to angiogenesis, invasion, metastasis, poor survival, and resistance to targeted therapies in CRC patients.²²³

All four FGFRs and their corresponding ligands are expressed in CRC.²²⁴ Among them, FGFR1 is frequently overexpressed in CRC patients and is associated with aggressive clinical behavior.²²⁵ Additionally, FGFR2 regulates CRC cell migration, invasion, and growth, playing a crucial role in cancer progression.

NTRK1, NTRK2, and NTRK3 receptors are activated by NGF, BDNF, and neurotrophic factor-3, respectively. These receptors play a role in the proliferative and migratory processes in the nervous system.²²

ROR1 has been identified as both a prognostic marker and a potential therapeutic target for CRC. It is frequently overexpressed in CRC cells compared with healthy tissues and shows a positive correlation with clinical stage and lymph node metastases.²²⁶ The overexpression of ROR1 in CRC cells relative to surrounding normal tissues suggests its potential as an indicator of prognosis and a target for therapy in CRC.

High levels of HGF have been recognized as a significant prognostic marker in CRC. This is because HGF has the ability to promote the proliferation, motility, adhesion, and invasion of CRC cells, and is closely associated with the development, progression, and spread of CRC.²²⁷ In CRC, elevated levels of HGF often coincide with the overexpression of the MET receptor. The MET proteins, in turn, activate various proteins such as survivin, the X-linked inhibitor of apoptosis protein, and other inhibitory apoptotic proteins through the AKT pathway, leading to tumor infiltration and distant metastasis.²²⁸

The AXL tyrosine kinase receptor is highly expressed in CRC^{229, 230} and is involved in various processes such as epithelial-to-mesenchymal transition, tumor angiogenesis, resistance to chemotherapy and targeted therapies, and suppression of the antitumor immune response.²³⁰ AXL is considered a potential therapeutic target for the treatment of CRC, especially in cases where adjuvant therapy targeting EGFR/VEGF has been ineffective.²³¹

During the early stages of malignant transformation in CRC, the expression of EphA1, EphA2, EphB1, EphB2, and EphB4 is increased, suggesting their potential involvement in tumor invasion and metastasis.²³² However, as CRC progresses, the expression of Eph proteins generally diminishes and eventually disappears in advanced CRC. Nonetheless, the expression of Eph proteins has been acknowledged as a potentially valuable marker in identifying individuals with CRC.²³²

In CRC, RET is considered a tumor suppressor kinase.²³³ The inactivation of the RET gene, due to abnormal methylation or mutation, may contribute to the progression of colonic adenomas into cancer.^{233, 234}

Besides the mentioned RTKs, several other receptors including VEGFR,^{235, 236} EGFR,^{227, 237} STYK1,²³⁸ ALK,^{239, 240} LMTK3,²⁴¹ TIE2,²⁴² ROS1,^{239, 240} IGF1R,²⁰ TYRO3,²⁴³ and MER²⁴³ have also been found to have prognostic significance in CRC.

6.1 Multitarget tyrosine kinase inhibitors (small molecules)

Crizotinib is an ATP-competitive multitarget protein kinase inhibitor that targets Met, ALK, and ROS1. It has shown promise in treating patients with ALK-positive NSCLC. However, patients treated with crizotinib typically develop resistance within a year or two, and there is a higher incidence of central nervous system (CNS) progression or metastasis in ALK-positive NSCLC patients who receive crizotinib as a first-line treatment. As a result, second-generation ALK inhibitors such as ceritinib, alectinib, brigatinib, and ensartinib, along with the third-generation lorlatinib, have emerged as alternatives after crizotinib resistance.²⁶⁰

Loratinib, a reversible and potent third-generation tyrosine kinase inhibitor, exhibits high selectivity by targeting ALK and ROS1. Notably, it has excellent penetration into the CNS and has shown effectiveness in treating patients with intracranial metastases after previous treatment with second-generation ALK inhibitors.²⁶¹ Several studies have demonstrated that patients treated with lorlatinib experience significantly longer progression-free survival and a higher occurrence of intracranial reactions compared with those treated with crizotinib.^{262, 263}

Cabozantinib, a broad-spectrum anticancer medication, has the ability to inhibit multiple targets including MET, VEGFR1/2/3, RET, KIT, FLT3, AXL, NTRK, ROS1, and others. Currently, it is considered as the first-line treatment for kidney cancer. Moreover, cabozantinib has shown remarkable efficacy in treating various other cancers such as thyroid cancer, liver cancer, soft tissue sarcoma, NSCLC, prostate cancer, breast cancer, ovarian cancer, and bowel cancer. In RCC, the combination of nivolumab and cabozantinib has demonstrated positive patient benefits.²⁶⁴

6.2 Specific tyrosine kinase inhibitors (small molecules)

Compared with first- and second-generation TKIs, osimertinib is a third-generation EGFR-positive lung cancer targeted medication that significantly improves patients’ overall survival and progression-free survival. In patients with EGFR-mutated stage IB-IIIA NSCLC, adjuvant osimertinib after full tumor excision dramatically increases DFS and reduces the risk of both local and distant recurrence.²⁶⁵ Osimertinib has also shown favorable patient benefit in combination with other drugs. Several trials have demonstrated that combining tepotinib and osimertinib could potentially overcome resistance caused by MET amplification and lead to a significant improvement in the objective remission rate for patients with MET-amplified NSCLC.^{260, 261}

Tepotinib, the first MET tyrosine kinase inhibitor approved for treating NSCLC, is currently being developed to treat solid tumors.²⁶⁶ The drug has demonstrated significant activity in subgroups of patients with varying ages, prior treatments, and brain metastases, while maintaining a reasonable safety profile and few treatment discontinuations.²⁶⁷

Tivantinib, a MET inhibitor, works by stabilizing the inactive conformation of the MET RTK. It prevents both constitutive and ligand-mediated activation through an ATP-independent binding mechanism. In patients with HCC who have failed sorafenib treatment, MET overexpression has been shown to be a negative prognostic indicator. In a second-line therapy phase II study for advanced HCC patients, those in the MET high expression group who were given tivantinib showed almost double the PFS and OS compared with those who received a placebo. Furthermore, the combination of tivantinib and sorafenib also showed promising results.²⁶⁸

Savolitinib is a MET inhibitor that has been developed to treat metastatic NSCLC, PRCC, gastric cancer, and CRC.²⁶⁹ Preclinical data suggest that the combination of EGFR TKIs and MET TKIs may be a possible treatment option for EGFR mutation-positive lung cancer with MET-driven acquired resistance. In patients with advanced NSCLC whose disease has progressed after previous use of an EGFR TKI and who have MET-amplification, the combination of savolitinib and osimertinib has shown promising antitumor activity with acceptable risk effects.²⁷⁰ In May 2020, the National Medical Products Administration of China accepted a new drug marketing application for the MET inhibitor savolitinib, intended for the treatment of NSCLC with MET exon 14 skipping. Savolitinib has been authorized as a novel medicine for both domestic and international markets.

SCC244 is a highly selective inhibitor of MET kinase that has demonstrated subnanomolar potency against MET kinase activity and excellent selectivity versus 312 other evaluated protein kinases. The administration of SCC244 has shown strong anticancer efficacy at well-tolerated levels in xenografts of human tumor cell lines or NSCLC and HCC patient-derived tumor tissue driven by MET abnormality. These findings provide a solid platform for the clinical examination of SCC244 in patients with malignancies that are altered along the MET pathway.²⁷¹ A study demonstrated that when SCC244 and HAI-2 were used together, they had a greater inhibitory effect on the proliferation of RCC cells compared with when either inhibitor was used alone.²⁷²

Capmatinib demonstrated significant antitumor activity in patients with advanced NSCLC characterized by MET exon 14 skipping mutations, particularly in those who had not received prior treatment. A clinical trial involving 364 patients evaluated the efficacy of capmatinib. Among patients with NSCLC and a MET exon 14 skipping mutation who had previously undergone one or two lines of therapy, an overall response rate of 41% (95% confidence interval [CI], 29–53) was observed. For patients who had not received any prior therapy, the overall response rate was 68% (95% CI, 48–84). The median duration of response was 9.7 months (95% CI, 5.6 to 13.0) for previously treated patients and 12.6 months (95% CI, 5.6 to could not be estimated) for treatment-naïve patients. However, patients with MET amplification, who had undergone prior treatment and had a gene copy number of less than 10, exhibited limited efficacy with an overall response rate of 7−12%.²⁷³

6.3 Antireceptor antibodies

The US FDA authorized cetuximab, a chimeric murine human monoclonal antibody of the IgG1 subclass, in February 2004 for the treatment of advanced CRC. It was the first EGFR mAb to be approved. Early phase II clinical trials of cetuximab showed promising results.²⁷⁴ According to a randomized phase II trial, patients who were irinotecan-refractory and received cetuximab + irinotecan had a median OS of 22.9 months (218 participants), while those who received irinotecan alone had a median OS of 10.8 months (111 subjects).²⁷⁵ However, the effectiveness and overall survival of cetuximab may be influenced by alterations in the KRAS gene. Currently, patients with KRAS wild-type and EGFR-positive metastatic CRC are treated with cetuximab and chemotherapy as their first-line treatment.

SAIT301 has been found to downregulate MET and effectively inhibit the growth of tumors with low or no Cbl expression. It also has the ability to prevent the growth of tumors with the deletion of MET exon 14, where MET binds to Cbl.²⁷⁶ According to a study, SAIT301 utilizes a MET degradation pathway that is mediated by LRIG1 and independent of Cbl. This pathway avoids the effect that has been known to limit the effectiveness of MET antibodies.²⁷⁶ SAIT301 has significant implications in the diagnosis and treatment of nasopharyngeal carcinoma. The inhibitory effect of SAIT301 on MET significantly reduces the migration and invasion of HNE1 cells. Additionally, SAIT301 can also decrease the nonanchored growth of HGF-induced HNE1 cell lines.²⁷⁷ A study found that the anticancer activity of SAIT301 was enhanced in MET-amplified MKN45 cells when 69 genes were knocked down.²⁷⁸ Further analysis of these genes revealed that FGF receptor (FGFR) is a crucial regulator of the antiproliferative effects of MET-targeted drugs. Integrin β3 is also a promising target for combination therapy with SAIT301. Gene expression analysis using the CCLE database suggests that FGFR and integrin β3 can be used as predictors of MET-targeted therapy,²⁷⁸ offering a potential therapeutic solution to overcome the acquired and innate resistance to MET-targeted drugs.

6.4 Antiligand antibodies

Bevacizumab is the first medication authorized for sale that targets tumor angiogenesis. It has been proven effective in treating various malignant tumors and is approved for multiple indications, including metastatic CRC, NSCLC, breast cancer, GBM, ovarian cancer, RCC, and cervical cancer. Its mechanism of action involves selectively targeting VEGF to exert antitumor effects.²⁷⁹ Combining chemotherapy with bevacizumab has shown benefits for patients. A study revealed that patients with refractory metastatic CRC who received FTD-TPI + bevacizumab treatment had longer overall survival compared with those who received FTD-TPI alone.²⁸⁰ Bevacizumab marked the beginning of a novel approach to anticancer therapy and remains the most widely used antiangiogenic treatment.

Rilotumumab is a monoclonal antibody that targets the HGF protein, preventing it from binding to MET. A study revealed that the effectiveness of rilotumumab is dependent on the dose given to patients with MET-positive gastric cancer or cancer of the esophagogastric junction.²⁸¹ However, another study found that inhibiting the MET pathway with rilotumumab did not improve the clinical prognosis of patients with MET-positive gastric or gastroesophageal adenocarcinoma.²⁸² Resistance is a common obstacle to effective cancer treatment, and acquired resistance is becoming a significant issue for targeted therapies. Rilolotuzumab resistance is acquired through unusual mechanisms, including dramatic HGF overproduction and misfolding, ER stress response signaling, and redirected vesicle trafficking.²⁸³

6.5 RTKs inhibitors in combination with immunotherapy

While some immunotherapies, including pericyte transfer (ACT) and immune checkpoint inhibitors, have shown promise in providing long-lasting clinical responses, their efficacy is not consistent and only a portion of cancer patients can benefit from them. The inflammatory response in the TME is a significant factor contributing to tumor progression and poor prognosis. However, accurately profiling immune cells within the TME has been limited due to its highly heterogeneous and dynamic nature. Fortunately, recent advancements in single-cell technologies such as single-cell RNA sequencing (scRNA-seq) and mass spectrometry flow cytometry have made it possible to systematically detect immune cells within the TME and gain new insights into their functional diversity.²⁸⁴

Patients with advanced MET 14 exon alterations in lung cancer show a long-lasting response to MET inhibitors. A study reveals that a considerable proportion of lung cancers with MET exon 14 alterations express PD-L1, but their median tumor mutation burden is lower than that of unselected NSCLCs.²⁸⁵ Although PD-1 blocking sometimes elicits responses, its overall clinical effectiveness is limited.

The interaction between MET and TME has been linked to secondary gliomas. However, the effect of MET genes on primary gliomas, specifically GBM, and their ability to evade immunosurveillance checkpoints is not well understood. A recent study proposes that the MET/STAT4/PD-L1 axis and tumor-associated macrophages may contribute to glioma immune evasion and lead to poor prognosis in GBM cases. This finding suggests a potential clinical approach for targeted therapy combined with immunotherapy for primary GBM patients.²⁸⁶

Pancreatic cancer is a highly malignant tumor with a complex immune microenvironment. Current RTK targeting strategies have limited effectiveness in treating this cancer. However, recent research has identified MET as a pancreatic cancer-specific RTK that is significantly associated with prognosis in both immunologically “hot” and “cold” pancreatic cancers. Studies have shown that MET is highly upregulated in pancreatic cancer tissues and positively correlated with PD-L1 levels. Additionally, elevated MET and PD-L1 expression have been strongly associated with lymph node metastasis, tumor TNM staging, and overall survival in pancreatic cancer. MET can interact with PD-L1 and help maintain its expression level in various ways. Lowering the expression of MET can increase the infiltration of lymphocytes into pancreatic tumors.²⁸⁷

To enhance the clinical benefits, targeted medicines can be combined with different inhibitors and immunotherapy for the treatment of cancers. Currently, several clinical trials are underway to investigate new targeted medications. The outcomes of some clinical trials for RTKs are summarized in Table 3.