Tyrosine kinase (TK) cascades are involved in all stages of tumorigenesis through modulation of transformation and differentiation, cell-cycle progression, and motility. Advances in molecular targeted drug development allow the design and synthesis of inhibitors targeting cancer-associated signal transduction pathways. Potent selective inhibitors with low toxicity can benefit patients with local and metastatic malignancies. This article evaluates information on solid tumor–related TK signaling and inhibitors, including receptor TK signal pathways that lead to successful application in clinical settings, properties of recently approved TK-inhibitor drugs for the treatment of solid tumors, and potential TK pathways for future therapeutic interventions.
Key points
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Hyperactivation of receptor tyrosine kinases (TK) is common in solid tumors.
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Inhibitors targeting TK signaling have been approved by the Food and Drug Administration to treat patients with primary and metastatic malignancies including cancers of the colon, lung, breast, pancreatic, and head and neck.
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Current advances in cancer-associated TK signaling, antibodies that block and neutralize receptor TK, and small-molecule drug targeting of TK signaling are evaluated and summarized.
Introduction
What are Tyrosine Kinase Inhibitors?
Animal tissues rely on cell-surface receptor tyrosine kinases (TK) to detect and respond to nutrients and growth factor hormones. Deregulated TK signaling, including overreaction to extracellular stimuli or autocrine activation, plays an important role in tumorigenesis. Hyperactivation of receptor TK caused by gene mutation, amplification, rearrangement, and transcriptional and translational deregulation is common in the tissues of solid tumors. Better understanding of receptor TK signaling in the modulation of cell transformation, proliferation, apoptosis, angiogenesis, invasion, and migration can help the design of inhibitors to treat malignancies.
The TK family consists of 58 receptor and 32 nonreceptor or cytoplasmic TK. Receptor TK span through the cell membrane with an extracellular and an intracellular domain. Stimuli such as growth factors bind to the extracellular domain, often leading to change and activation of TK conformation. The cytosolic kinase domain of TK catalyzes the transfer of a phosphor group from adenosine triphosphate (ATP) to the tyrosine residues of substrate proteins. The chain activation of cascade kinases transmits microenvironmental cues to cellular growth, survival, or death. Deregulation of the outside-in signaling contributes to the abnormal survival and/or uncontrolled growth in tumorigenesis.
Inhibitors are designed to target specific receptor TK that are overactive because of insult-induced protein damage and/or genetic mutation-caused loss/gain of function in tumors. In comparison with classic cytotoxic chemotherapy, targeted treatment is expected to have fewer side effects or normal tissue injury, but higher selectivity or potency in inducing cancer cell death. Disadvantages of targeted therapies are: (1) the molecular, biochemical, and cellular information of dysfunctional TK must be available before TK inhibitors (TKI) can be designed ; (2) the outcomes of the targeted therapy can vary among patients because the signal alternations are varied even in the same tumor ; and (3) the effects of off-target inhibition can cause side effects.
Current advances in novel technologies, disease models, and genetic manipulations allow identification of tumor-related signal pathways. TKI targeting such pathways have been developed. Searching the National Institutes of Health clinical trial database ( clinicalTrial.gov ) using the terms “tyrosine kinase inhibitors and cancer,” 448 hits were revealed. Major targets include the epidermal growth factor receptor (EGFR), the human epidermal growth factor receptor 2 (HER2), the platelet-derived growth factor receptor (PDGFR), the vascular endothelial growth factor receptor (VEGFR), the fibroblast growth factor receptor (FGFR), c-Met (MET or MNNG HOS transforming gene) or the hepatocyte growth factor receptor (HGFR), and the insulin-like growth factor receptor (IGF-1R). Growth factor ligands bind and activate the receptor TK, inducing downstream signal transduction pathways including PI3K/Akt/mTOR, RAS/RAF/MEK/ERK, JAK/FAK/Src/STAT, and PLC/DAG/PKC signaling ( Fig. 1 ). These pathways modulate cell-cycle progression, survival, differentiation, migration, and apoptosis. Deregulation caused by mutation, amplification, and overexpression or suppressed expression contributes to tumorigenesis.
Introduction
What are Tyrosine Kinase Inhibitors?
Animal tissues rely on cell-surface receptor tyrosine kinases (TK) to detect and respond to nutrients and growth factor hormones. Deregulated TK signaling, including overreaction to extracellular stimuli or autocrine activation, plays an important role in tumorigenesis. Hyperactivation of receptor TK caused by gene mutation, amplification, rearrangement, and transcriptional and translational deregulation is common in the tissues of solid tumors. Better understanding of receptor TK signaling in the modulation of cell transformation, proliferation, apoptosis, angiogenesis, invasion, and migration can help the design of inhibitors to treat malignancies.
The TK family consists of 58 receptor and 32 nonreceptor or cytoplasmic TK. Receptor TK span through the cell membrane with an extracellular and an intracellular domain. Stimuli such as growth factors bind to the extracellular domain, often leading to change and activation of TK conformation. The cytosolic kinase domain of TK catalyzes the transfer of a phosphor group from adenosine triphosphate (ATP) to the tyrosine residues of substrate proteins. The chain activation of cascade kinases transmits microenvironmental cues to cellular growth, survival, or death. Deregulation of the outside-in signaling contributes to the abnormal survival and/or uncontrolled growth in tumorigenesis.
Inhibitors are designed to target specific receptor TK that are overactive because of insult-induced protein damage and/or genetic mutation-caused loss/gain of function in tumors. In comparison with classic cytotoxic chemotherapy, targeted treatment is expected to have fewer side effects or normal tissue injury, but higher selectivity or potency in inducing cancer cell death. Disadvantages of targeted therapies are: (1) the molecular, biochemical, and cellular information of dysfunctional TK must be available before TK inhibitors (TKI) can be designed ; (2) the outcomes of the targeted therapy can vary among patients because the signal alternations are varied even in the same tumor ; and (3) the effects of off-target inhibition can cause side effects.
Current advances in novel technologies, disease models, and genetic manipulations allow identification of tumor-related signal pathways. TKI targeting such pathways have been developed. Searching the National Institutes of Health clinical trial database ( clinicalTrial.gov ) using the terms “tyrosine kinase inhibitors and cancer,” 448 hits were revealed. Major targets include the epidermal growth factor receptor (EGFR), the human epidermal growth factor receptor 2 (HER2), the platelet-derived growth factor receptor (PDGFR), the vascular endothelial growth factor receptor (VEGFR), the fibroblast growth factor receptor (FGFR), c-Met (MET or MNNG HOS transforming gene) or the hepatocyte growth factor receptor (HGFR), and the insulin-like growth factor receptor (IGF-1R). Growth factor ligands bind and activate the receptor TK, inducing downstream signal transduction pathways including PI3K/Akt/mTOR, RAS/RAF/MEK/ERK, JAK/FAK/Src/STAT, and PLC/DAG/PKC signaling ( Fig. 1 ). These pathways modulate cell-cycle progression, survival, differentiation, migration, and apoptosis. Deregulation caused by mutation, amplification, and overexpression or suppressed expression contributes to tumorigenesis.
TK signaling and solid tumors
EGFR Signaling in Proliferation and Migration of Cancer Cells
Function
EGFR belongs to a closely related ErbB subfamily that consists of EGFR (ErbB-1), HER2/c-neu (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4). The gene symbol ErbB is derived from erythroblastic leukemia viral oncogene, to which these receptors are homologous. EGFR is located across the cell membrane, and its surface portion binds to extracellular protein ligands including epidermal growth factors (EGF) and transforming growth factor α. Ligand binding triggers the conformation change and dimerization of EGFR, leading to TK activity of its cytosolic domain. EGFR autophosphorylation at multiple sites such as Y992, Y1045, Y1068, Y1148, and Y1173 elicits downstream signaling through tyrosine phosphorylation of effectors including mitogen-activated protein kinase (MAPK), Akt, and JNK. EGFR activation induces DNA synthesis and suppresses apoptosis, contributing to cell migration, adhesion, and proliferation (see Fig. 1 ).
Tumorigenicity
Genetic mutations and/or insults that promote EGFR ligand binding, sustained activation, and receptor TK phosphorylation can enhance cell survival and abnormal growth. Overexpression, deregulation, and hyperactivation of EGFR or its family members are associated with cancers that are derived from epithelium. Identification of oncogenic EGFR has attracted great attention in the attempt to develop therapeutic interventions to treat EGFR hyperactive tumors. Receptor-neutralizing antibodies and small molecules are used to prevent ligand interactions, conformation changes or activation, and ATP binding.
HER2 and Cell Growth
HER2 is encoded by the ERBB2 gene and belongs to the EGFR family. There is no known ligand that binds to HER2. It is a preferred binding partner of other EGFR members in forming heterodimers. Dimerization induces the autophosphorylation of the cytosolic kinase region, triggering several cascade events such as MAPK, PI3K/Akt, PPCγ, PKC, and STAT signal-transduction pathways (see Fig. 1 ).
EGFR signaling potently promotes cell proliferation and inhibits apoptosis. Hyperactivity of HER2 contributes to uncontrolled growth in tumorigenesis. Overexpression and/or alterations of HER2 have been observed in breast, ovarian, stomach, and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma. HER2 inhibition can prolong the survival of patients with cancer.
VEGFR and Tumor Angiogenesis
Uncontrolled cell proliferation increases tumor size, leading to oxygen and nutrient deficiency due to perfusion limitations. Under hypoxic or nutrient-depleted conditions normal cells will die, but malignant cells live and survive. One of the adaptive responses of tumor cells is to release the vascular endothelial growth factor (VEGF). Endothelial cells line the blood vessels. The very end of the vasculature (capillary) consists of a single layer of endothelial cells that express VEGFR. In responding to VEGF, the endothelium expands or sprouts to form new vessels/branches to supply oxygen and nutrients.
VEGF binding to its receptor induces VEGFR dimerization and activation through transphosphorylation. VEGF-A interacts and activates VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). VEGF-C and VEGF-D bind to VEGFR3. VEGFR2 is responsible for most VEGF-stimulated cellular responses. VEGFR1 seems to modulate VEGFR2 action as a decoy. A soluble form of VEGFR1 can be secreted, and serves as a decoy through binding to VEGF-A. VEGFR3 plays a role in lymphangiogenesis. TKI are designed to interrupt the VEGF binding, VEGFR transphosphorylation, and substrate-effector activation (see Fig. 1 ). Preventing VEGF stimulation of angiogenesis or forming new blood vessels can cut off oxygen and nutrient supplies, often stopping tumor growth. However, some cancer cells can develop resistance to VEGFR inhibition through activation of alternative pathways.
PDGFR and Cell Differentiation and Proliferation
Phenotype changes are among the major hallmarks of malignancies. PDGFs play key roles in cell differentiation, proliferation, and growth. The PDGF family consists of PDGF-A, PDGF-B, PDGF-C, and PDGF-D, which can form homodimers or heterodimers: AA, BB, AB, CC, or DD. PDGFR-α and PDGFR-β are PDGF receptors encoded by different genes. On PDGF stimulation, the PDGFR can form αα, ββ, or αβ dimers. The combinations among PDGF and their receptors are varied in different cell types. Dimerization/transphosphorylation induces the PDGFR conformation change, resulting in kinase activation. Phosphorylation of downstream factors induces signaling cascades including RAS/MAPK and PI3K/PLCγ pathways (see Fig. 1 ). PDGFR overexpression and/or hyperactivity are common in solid tumors. For instance, the gene copy numbers of PDGFR are increased in non–small cell lung carcinoma (NSCLC).
FGFR and Tumorigenesis
Four genes encode FGFR1, FGFR2, FGFR3, and FGFR4. Natural alterations of transcriptional splicing can generate 48 isoforms of FGFR. There are 22 fibroblast growth factors (FGF), belonging to the largest family of growth factor ligands. Each ligand can bind to several FGFR that vary in properties. FGFR-mediated signaling contributes to cell proliferation, differentiation, survival, and invasion (see Fig. 1 ). Protein alterations, upregulation, and fusion caused by genetic mutation and rearrangements of FGFR genes are associated with many types of malignancies including prostate, lung, sarcoma, breast, bladder, melanoma, and moderately/poorly differentiated endometrial cancer. Fewer studies have been performed on the development of TKI targeting FGFR in comparison with TKI targeting EGFR, VEGFR, PDGFR, and IGF-1R. Challenges are related to complexity of FGFR signaling pathways, the presence of fibroblasts in normal and tumor tissues, and a weak correlation between FGFR hyperactivity and tumor prognosis.
c-Met and Invasive Growth
The HGFR protein is encoded by c-Met. Posttranslational modifications of the precursor protein include cleavage and disulfide linking, resulting in the mature receptor. Hepatocyte growth factor is the only known ligand for HGFR, which is produced and released by cells of mesenchymal origin. HGFR is expressed in the cells of epithelial origin. On stimulation of hepatocyte growth factor, HGFR phosphorylates/activates downstream factors including PLCγ/PKC, PI3K/Akt, Ras/Raf/ERK, and FAK/Src (see Fig. 1 ). The activated cascades modulate invasive growth to generate new organs and tissues in embryonic development and tissue-wound healing.
Cancer cells can hijack the HGFR-related growth process for their invasion and metastasis. C-Met overexpression contributes to apoptotic resistance of pancreatic and ovarian cancer. Searching the ClinicalTrial.gov Web site (February 2013) revealed 10 studies on TKI targeting c-Met in advanced solid tumors, including lung and gastric cancer. Completion of these studies will help identify novel drugs to treat invasive and metastatic tumor.
IGF-1R and Tissue Overgrowth
The IGF-1R precursor is cleaved into 2 peptides (α and β subunits) and then linked via disulfide bonds to form a mature receptor. Its ligands, IGF-1 and IGF-2, are growth hormones with structural similarity to insulin, and bind to insulin receptors with low affinity. IGF-1R signaling promotes cell growth and tissue hypertrophy (see Fig. 1 ). Epithelial cells proliferate and expand to form duct and gland structures. After weaning, the epithelium disappears through apoptosis. In this process, IGF-1R is believed to promote differentiation and inhibits apoptosis.
Activation of IGF-1R signaling is correlated with primary and metastatic malignancies such as prostate, breast, pancreatic, and lung cancer. The antiapoptosis property of IGF-1R–triggered kinase cascades contributes to cancer-cell resistance to cytotoxicity and vascularization. When EGFR pathways are blocked by inhibitors such as erlotinib, IGF-1R can resume EGFR-related signaling and promote survival in breast cancer. IGF-1R–enhanced angiogenesis is involved in tumor invasion and metastasis.
Increased glucose uptake and metabolism are hallmarks of malignancies. Abnormal IGF-1R signaling can contribute to the tumor metabolism in several ways. First, IGF-1 binds to the insulin receptor, stimulating glucose uptake. Second, IGF-1R is associated with the insulin receptor. Hyperactivity of IGF-1R may enhance insulin receptor–induced glucose metabolism. Finally, IGF-1R signaling may promote the shift of cellular metabolism toward the generation of building materials to support proliferation. TKI targeting IGF-1R signaling can prevent abnormal glucose uptake and aerobic glycolysis.
c-Kit and Cell Proliferation
Tyrosine protein kinase kit (c-Kit) or CD117 is the cellular homology of the feline sarcoma viral oncogene v-kit. Hematopoietic stem cells, mast cells, skin melanocytes, and interstitial cells express c-Kit on the cellular surface. On binding to the stem-cell factor, c-Kit forms dimers and phosphorylates/activates effector proteins, modulating cell survival, proliferation, and differentiation.
The activity and levels of c-Kit are increased in several types of solid tumors such as gastrointestinal stromal tumor, seminoma, breast cancer, and lung adenocarcinoma. For example, gain-of-function mutation of the KIT gene causes constitutive activation of the receptor even in the absence of its ligand, stem-cell factor, in human gastrointestinal stromal tumors. Specific inhibitors have been developed to target hyperactivated c-Kit in these tumors, which marks the new era of targeted molecular therapy. The TK inhibitor Gleevec (imatinib mesylate), has been approved by the Food and Drug Administration (FDA) to treat patients with tumors such as Philadelphia chromosome–positive chronic myeloid leukemia and KIT-positive unresectable and/or metastatic malignant gastrointestinal stromal tumors.
Other Inhibitor-Targeted TK
The TK family consists of numerous members that are associated with tumorigenesis. Many TK have been studied for the development of inhibitors to treat malignancies according to the National Institutes of Health clinical trials database: estrogen receptor (ER), anaplastic lymphoma kinase (ALK), colony-stimulating factor 1 receptor (CSF1R), Raf/MAPK, PI3K/Akt, mTOR, JAK, and HSP90; they can be classified into receptor (ER, ALK, CSF-1R) and cytosolic (Raf/MAPK, PI3K/Akt, mTOR, JAK, and HSP90) TK families. Most cytosolic TK are downstream effectors of receptor TK. Overexpression, amplification, and fusion of the effector TK can result in their constitutive activation even in the absence of growth-factor stimulation. TKI targeting the effector kinases can block malignancy-associated overgrowth. However, the effector cascades are often shared by multiple receptor TK in responding to a variety of extracellular stimulation; they are essential for normal cell function, survival, and growth. TKI targeting downstream factors can be toxic to normal cells and can cause strong side effects.
New TKI drugs approved by the FDA
A molecular targeted therapeutic approach has the great advantage of specific inhibition of survival pathways in cancer. Intensive pharmaceutical research and development of TKI have led to the release of novel anticancer drugs. New TKI drugs, particularly FDA-approved anticancer TKI for solid tumors, are reviewed in this section. Basic information such as inhibitor types, targets, and FDA indications are summarized in Table 1 . The most significant examples of new drugs targeting a particular pathway are briefly described.
| Name | Trade Name | Manufacturer | Type | Target | FDA Indication | Date |
|---|---|---|---|---|---|---|
| Cetuximab | Erbitux | Bristol-Myers Squibb & Eli Lilly | Mouse/human monoclonal antibody | EGFR | Squamous-cell carcinoma of the head and neck | 2006, 2008 |
| KRAS wild-type metastatic colorectal cancer | 2009 | |||||
| Therascreen KRAS test | 2012 | |||||
| Panitumumab | Vectibix | Amgen | Human monoclonal antibody | EGFR | EGFR-expressing metastatic colorectal cancer | 2006 |
| Relabeled for including KRAS detection | 2009 | |||||
| Trastuzumab emtansine | Kadcyla | Genentech/Roche | Antibody and toxin conjugate | HER2 | HER2-positive metastatic breast cancer | 2013 |
| Pertuzumab | Perjeta | Genentech/Roche | Human monoclonal antibody | HER2 | HER2-positive metastatic breast cancer | 2012 |
| Bevacizumab | Avastin | Genentech/Roche | Human monoclonal antibody | VEGFR | Colon cancer | 2004 |
| Lung cancer | 2006 | |||||
| Breast cancer | Approved 2008, revoked 2011 | |||||
| Renal and brain cancer | 2009 | |||||
| Lapatinib | Tykerb/Tyverb | GSK | N -[3-Chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[(2-methylsulfonylethylamino)methyl]-2-furyl]quinazolin-4-amine | HER2, EGFR | Breast cancer | 2007 |
| HER2-positive metastatic breast cancer | 2010 | |||||
| Cabozantinib | Cometriq | Exelixis | N -(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)- N -(4-fluorophenyl)cyclopropane-1,1-dicarboxamide | c-Met, VEGFR2 | Medullary thyroid cancer | 2012 |
| Regorafenib | Stivarga | Bayer | 4-[4-({[4-Chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]- N -methylpyridine-2-carboxamide hydrate | VEGFR2, Tie2 | Metastatic colorectal cancer | 2012 |
| Advanced gastrointestinal stromal tumors | 2013 | |||||
| Axitinib | Inlyta | Pfizer | N -Methyl-2-[[3-[(E)-2-pyridin-2-ylethenyl]-1H-indazol-6-yl]sulfanyl]benzamide | VEGFRs, PDGFR | Renal cell carcinoma | 2012 |
| Aflibercept | Zaltrap | Sanofi-Aventis and Regeneron Pharmaceuticals | VEGF-A/-B, PIGF | Metastatic colorectal cancer | 2012 | |
| Crizotinib | Xalkori | Pfizer | 3-[(1R)-1-(2,6-Dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine | Non–small cell lung carcinoma | 2011 | |
| Vemurafenib | Zelboraf | Plexxikon/Roche | B-Raf V600E | Late-stage melanoma | 2011 | |
| Sunitinib | Sutent | Pfizer | N -(2-Diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide | PDGFR, VEGFR, c-Kit, Ret, CSFR, flt3 | Renal and gastrointestinal stromal tumors | 2006 |
| Pancreatic neuroendocrine tumors | 2011 | |||||
| Vandetanib | Caprelsa | AstraZeneca | N -(4-Bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine | EGFR, VEGFR, RET-TK | Metastatic medullary thyroid cancer | 2011 |
| Everolimus | Zortress | Novartis | Dihydroxy-12-[(2R)-1-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone | mTOR | Advanced kidney cancer | 2009 |
| Subependymal giant-cell astrocytoma | 2010 | |||||
| Metastatic pancreatic neuroendocrine tumors | 2011 | |||||
| Breast cancer | 2012 | |||||
| Erlotinib | Tarceva | Genentech/Roche | N -(3-Ethynylphenyl)-6,7-bis(2-methoxyethoxy) quinazolin-4-amine | EGFR | Lung cancer | 2004, 2009 |
| Pancreatic cancer | 2009 |
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