Introduction

It has taken almost a century to understand the biological function of Src, as well as its structure, activation, and regulatory network. In the 1910s, Peyton Rous proposed a hypothesis that solid tumors are caused by infection of a 'filter agent'. In the 1950s, the Rous sarcoma virus (RSV) was shown to cause tumors and alter the morphology of chicken embryo fibroblasts.1 Once the v-Src gene was sequenced and information about its signaling properties was discovered in the 1970s, c-Src was shown to be a 'proto-oncogene' in normal mammalian cells. In 1989, the Nobel Prize in Physiology and Medicine was awarded for this discovery, which was described as 'the cellular origin of retroviral oncogenes'.2,3 In the 1980s, the Src homology 2 domain (SH2 domain) was found to be a selective domain that recognizes phosphorylated tyrosine residues.4 Src homology 3 domains (SH3 domains) were sequenced and identified in c-Src in 1993; these domains mediate protein–protein interactions via binding to proline-rich peptides.5 Finally, the three-dimensional structure was revealed in the 1990s, allowing a better understanding of the structure and biological functions of c-Src.6,7

c-Src belongs to a family of nonreceptor tyrosine kinase proteins and is ∼60 kDa in size. As a cytoplasmic protein, c-Src has a critical role in mediating signal transduction via interactions with multiple proteins and protein complexes. The aberrant activation of c-Src regulates multiple functions during tumor progression, such as apoptosis, proliferation, cell adhesion, cell migration and invasion, angiogenesis, and metastasis. The nine members of the Src-family kinases (SFKs) are c-Src, Yes, Fyn, Lyn, Lck, Hck, Fgr, Blk, and Yrk;8 these members share a similar structure, all having SH1 (catalysis domain), SH2, SH3, and SH4 (Src homology domain 4) domains. Different tissues vary widely in expression levels of SFKs,9 and clinical studies have shown that aberrant activation of c-Src is correlated with malignant progression of cancer; high expression levels of c-Src are found in a number of human tumors, including lung, breast, pancreatic, colon, and prostate cancers.9,10,11,12

Structure and activation of c-Src

The structure of c-Src protein is composed of seven parts: SH4 domain, unique domain, SH3 domain, SH2–SH3 linker, SH2 domain, SH1 (catalytic domain), and C-terminal negative regulatory region. The SH4 domain contains a myristoylation sequence, which is important for membrane localization.2,6 The SH3 domain can bind to proline-rich peptide binding sites and thus is important for protein–protein interactions.13 SH2 domains were first identified as protein motifs that bind phosphorylated tyrosine sites.2 The SH1 domain contains the catalytic kinase domain as well as tyrosine 419, which is autophosphorylated by active c-Src.6 The C-terminal negative regulatory region harbors Y530, which can interact with the SH2 domain when phosphorylated by C-terminal Src kinase.14

Activation and inactivation of c-Src is accompanied by conformational changes in the protein (Figure 1). In the inactivated state, the SH2 domain recognizes and binds to the phosphorylated Y530 residue of the C-terminal of c-Src,15 producing a 'closed' state that prevents interaction of substrate proteins with the kinase domain. c-Src activity and configuration changes can be affected by multiple factors, such as SH2-binding and SH3-binding ligands, activation of the catalytic domain, or dephosphorylation of the C-terminal region at Y530.6 Dephosphorylated Y530 at the C-terminal region induces the C-terminal region to disassociate from the SH2 domain, resulting in an 'open', activated state, which allows substrate protein access to the catalytic kinase site in the SH1 domain.16 Activation of receptor tyrosine kinases allows the phosphorylated tyrosine residues to compete for c-Src SH2 domains and activate the kinase. Genomic loss of Y530 in v-Src has been shown to cause continuously high activation levels and induction of tumorigenesis.8 Deletion of the C-terminus of the mutated c-Src, as reported in rare cases of colon cancer, contributes a tumorigenic effect similar to that performed by v-Src.17 Overexpression of the receptor-like protein tyrosine phosphatase-α can activate c-Src by dephosphorylation at Y530, which suggests that protein tyrosine phosphatase might be involved in tumorigenesis by activation of c-Src.18

Figure 1: Structure and activation of c-Src.
figure 1

The c-Src protein is composed of four Src homology domains (SH1, SH2, SH3, and SH4). The SH4 is located on the N-terminal region and contains a myristoylation sequence important for membrane attachment. The SH3 domain is a proline-rich target domain that binds and interacts with other proteins or with c-Src itself. The SH2 domain can recognize phosphorylated tyrosine-containing residues, critical for regulation of Src activity or binding to other tyrosine proteins. The SH2-linker, located between SH2 and SH1, can regulate Src activity and interact with SH3. The SH1 domain contains the kinase domain as well as tyrosine residue Y419, which is phosphorylated by the kinase domain. Src activity is regulated by the C-terminal tyrosine site Y530 interacting with the SH2 domain. When Y530 is phosphorylated by C-terminal Src kinase, the C-terminus binds to the SH2 domain and inhibits Src activation via changing the configuration of SH2, SH3, and SH2-linker sites; the entire configuration becomes locked and inactive. Dephosphorylation of Y530 by protein tyrosine phosphatases allows the Src SH1, SH2 and SH3 domains to become open and therefore fully activated.

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Regulation of cell adhesion and motility

SFKs can have important roles in cell signaling by interacting with numerous upstream and downstream signaling components (Figure 2). Focal adhesion can be described as cell attachment to the extracellular matrix by integrins or intercellular transmembrane receptors, which connect with multiple proteins, including GTPases, actin cytoskeleton, and focal adhesion kinase (FAK). Adherens junctions are needed for cell–cell connections, which mainly involve the cadherin–catenin mediator complex. Both processes are necessary for cell attachment and motility, and are important in the regulation of multiple functions, including cell proliferation and survival.19 c-Src can suppress the integrins attached to the extracellular matrix via phosphorylation of integrin subunits.20,21 c-Src can also interrupt RhoA function, which has an important role in actin filament assembly and stabilization of focal adhesion.22 c-Src activates FAK, R-Ras, and phosphatidylinositol phosphate kinase, which indirectly affect integrin–actin cytoskeleton assembly. Destabilization of focal adhesion is needed for cell motility.19

Figure 2: SFK signaling pathways and function.
figure 2

SFKs are involved in a number of major functions and signaling pathways. a | SFKs can interact with RTK and cooperate in activation of downstream signaling, leading to cell proliferation, survival, invasion and angiogenesis. SFKs cooperate with RTK-dependent activation of the Ras/Erk/MAPK pathway, enhancing DNA synthesis and cell proliferation. SFK activation also regulates P13K/Akt signaling to promote cell survival. Acting through transcriptional factors, such as STAT3 for example, SFKs promote the transcription and secretion of proangiogenic growth factors and cytokines such as VEGF and IL-8. b | SFKs can have critical roles in the control of cell adhesion, migration, invasion and metastasis. Activated SFKs can phosphorylate p120 catenin and disrupt adherens junction stabilized by E-cadherin. FAK is a critical downstream target of SFK that promotes invasiveness of cells. Targets downstream of SFK and FAK include p130Cas, paxillin and Rho A that form complexes with integrin molecules important in interactions with the extracellular matrix. Abbreviations: FAK, focal adhesion kinase; IL-8, interleukin 8; PI3K, phosphoinositide 3-kinase; RTK, receptor tyrosine kinase; SFK, Src family kinase.

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Regulation of cell invasion and survival

The adherens junction serves as a bridge that connects the actin cytoskeleton to neighboring cells through direct interaction, and maintains tissue renewal function in epithelial tissues.23 E-cadherin is believed to be a tumor suppressor that prevents cell invasion. During tumor development, loss of E-cadherin function in epithelial cells results in cells with enhanced invasive and metastatic ability. This process is called epithelial–mesenchymal transition. Constitutively activated c-Src can phosphorylate cadherin, resulting in loss of cadherin–catenin complex function, thereby promoting cell differentiation and invasiveness.24,25 In addition, loss of or defective SFK function impairs adherens junctions, which suggests that SFKs are critical signaling proteins for regulation of the adherens junction network.26 Activated c-Src also promotes FAK phosphorylation, which interrupts E-cadherin.27 Phosphorylation of p120 catenin by SFKs is accompanied by disruption of E-cadherin, resulting in enhanced cell migration and invasion in tumor cells.28 Aberrant activity of c-Src can cooperate with receptor tyrosine kinases to phosphorylate p120 and interrupt the adherens junction.27 Thus, SFKs can be critical in mediating epithelial–mesenchymal transition and tumor metastasis.

As an important regulator of cell invasion, FAK can cooperate with c-Src to form a complex to regulate cell adhesion, adherens junctions, and survival by adjusting integrin attachment. The resulting Src–FAK complex affects multiple proteins, such as actin cytoskeletal proteins, integrins, and the Rho family GTPases. The mechanisms of this signaling complex are complicated. For example, Src and FAK can affect cell migration by suppressing RhoA activity or by activating Ras.29 These two opposite processes illustrate the importance of c-Src regulation in the disassembly and assembly of actins for cell mobility. p130Cas and paxillin, both SFK substrates, are other important cell migration mediators that can be recruited and bound to FAK. FAK–Src–p130Cas contributes to cell invasive abilities via activation of matrix metalloproteinases 2 (MMP-2) and MMP-9.30 The activated Src–FAK complex also promotes cell survival via activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and enhances cell survival.31 PI3K/Akt can be activated by transformation of v-Src and can affect cell survival.32 The regulatory domain of PI3K (p85) contains an SH2 domain that can interact with phosphorylated tyrosine kinases, including FAK and Src; however, the mechanism of Src/PI3K is complex and requires further investigation.

Cell proliferation and survival

c-Src can interact bidirectionally with multiple tyrosine kinase receptors via its SH2 domain and trigger a cascade of downstream signaling as well as modification of the cooperating receptor. These cooperating receptors include EGFR, VEGF receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), insulin-like growth factor 1 receptor (IGFR-1), hepatocyte growth factor receptor, colony-stimulating 1 receptor, stem cell factor receptor, muscle-specific kinase, and others.33 EGFR can bind to c-Src and phosphorylate tyrosine sites on its C-terminal loop. In certain cellular conditions c-Src is required for EGF-induced proliferation. Overexpression of c-Src can increase EGF-induced DNA synthesis in cancer cells.34 c-Src can directly bind to EGFR and phosphorylate the Y845 residue, resulting in increased MEK and MAPK activity and enhanced cell mitogenesis and transformation.35 Coexpression of EGFR and c-Src leads to cell hyperproliferation and enhances the tumor migratory and invasion behaviors in breast cancer cells.36 c-Src can also interact with ErbB2/ErbB3 signaling, forming a heterocomplex with these receptors, which can contribute to enhanced biological functions by stabilization of ErbB2/ErbB3 signaling.37 c-Src, Fyn, and Yes are activated by PDGFR via its two autophosphorylated tyrosine sites (Y579 and Y581) in the PDGFR juxtamembrane region. Mutation of these two tyrosine phosphorylation sites can repress PDGF-induced c-Src activation and cause loss of binding ability of c-Src to the receptor.38,39 Furthermore, PY934 in PDGFR can also be phosphorylated by c-Src, and mutation of this site will repress PDGF-induced mitogenesis.40 Thus, SFK can cooperate with receptor tyrosine kinase to mediate important signaling cascades in cancer.

Regulation of angiogenesis

Angiogenesis, the generation of new blood vessels, is considered a critical hallmark in tumor development. Angiogenesis is a multistep process that includes endothelial cell migration, invasion, and proliferation.41 When angiogenic growth factors or cytokine ligands bind to endothelial cell receptors, the receptor is activated, triggering a cascade of downstream signaling events that enhance cell proliferation and migration and the formation of blood vessels. These angiogenic growth factors include VEGF, fibroblast growth factor (FGF), hepatocyte growth factor, angiopoietins, and interleukin (IL)-8. SFKs are involved in regulation of angiogenesis in two ways (Figure 3).

Figure 3: Tumor and stromal cell angiogenesis processes are regulated by SFKs.
figure 3

SFKs can have important roles in regulating angiogenesis both at the level of tumor cells and in stromal cells such as endothelial cells. Activated SFKs in tumor cells can promote the expression and secretion of angiogenic growth factors and cytokines, such as VEGF and IL-8. These growth factors can interact with their receptors on endothelial cells that recruit and activate SFK. For example, activation of the VEGF receptor leads to recruitment and activation of c-Src that promotes endothelial cell proliferation and migration and angiogenesis. SFK inhibitors can affect angiogenesis by i) inhibiting the expression of angiogenic growth factors (VEGF, IL-8) in tumors cells and ii) inhibiting downstream signaling elicited by angiogenic growth factors acting upon their cell surface receptors on endothelial cells. Abbreviations: IL-8, interleukin 8; SFK, Src family kinase.

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First, SFKs can control expression of angiogenic growth factors and cytokines by regulating their gene expression.42 SFKs can induce upregulation of VEGF gene expression.42,43 Repression of c-Src can downregulate VEGF expression and result in decreased tumor vascular formation in vivo.44 The angiogenic factor IL-8 (IL-8/CXCL8) is synthesized in tumor cells and has an important role in regulating angiogenesis.45 Expression of IL-8 is correlated with Src activation, and c-Src inhibition can reduce IL-8 expression levels in pancreatic cancer cells.46 Src inhibitors, such as PP1 and PP2, can repress IL-8 promoter activities, whereas activated v-Src can enhance IL-8 promoter activation in human aortic endothelial cells.47 Interestingly, IL-8 phosphorylates VEGFR-2 and forms a complex with c-Src. Treatment with a Src inhibitor can block IL-8-induced phosphorylation of VEGFR-2, Src-receptor complex formation, and vascular permeability.48

Second, SFKs can cooperate with angiogenic growth factor receptors, such as VEGFR, to elicit signaling in endothelial cells or tumor cells.42 Phosphorylated Src induced by VEGF can promote formation of the FAK/α (v) β5 signaling complex, which is required for vascular permeability response and neovascularization. In c-Src-deficient mice, there was a reduced vascular permeability response to VEGF, which indicates that c-Src is required for VEGF-induced angiogenesis.44 c-Src has also been shown to interact with VEGFR in endothelial cells as well as tumor cells. The VEGFR-1/SFK complex can lead to phosphorylation of FAK, p130Cas, and paxillin in colon cancer cells, and silencing c-Src results in reduced cell migration.49 Src is required for either VEGF-induced ERK1/2 or FAK activation, which leads to increased cell proliferation. Inhibition of Src kinase activities can suppress cell proliferation and migration in human umbilical vein endothelial cells.50

Preclinical studies with Src inhibitors

Disruption of the c-Src regulatory intramolecular site by site-directed mutagenesis leads to deregulation of kinase activity. Mutations in c-Src are not the predominant mechanism of SFK activation in human cancers; therefore, inhibiting a single target of Src is unlikely to be successful. PD180970, SU6656/SU6566, PP2, and PP1 are common Src inhibitors that have been widely used in preclinical studies. Dasatinib, AZD0530, and SKI-606 are all dual specific Src/ABL kinase inhibitors that are undergoing clinical testing (Table 1).

Table 1 Selected current trials with bosutinib, AZD0530 and dasatinib*
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PP1, an SFK tyrosine inhibitor, has been used since 1996. PP1 has been reported to selectively inhibit SFKs in vitro in receptor-induced T-cell activation.51 The combination of PP1 and gefitinib has shown enhanced effects on EGFR-dependent tumors, and this strategy has been successfully used in clinical trials with dasatinib and erlotinib.52,53 PP2, another common inhibitor, has also shown antiangiogenic effects by decreasing IL-8 expression.46 PD180970 can suppress cell growth and survival via inhibition of Src in multiple cancers, such as lung, melanoma, and colon.54,55,56 SU6656 can block PDGFR- or IGFR-induced Src activities. PD180970 and SU6656 can inhibit PY-STAT3 (705), which is a critical phosphorylated tyrosine site in STAT3.54,57,58 The dual specific Src/ABL inhibitor, dasatinib, is a potent inhibitor of imatinib-resistant mutant cell lines, which specifically blocks Lyn and Src, and suppresses cell adhesion, migration, and invasion in human prostate cancer cells.59 In an in vivo pancreatic tumor study, dasatinib was shown to decrease tumor size and reduce metastases by blocking Src activation.60 Our group has also shown that dasatinib-induced apoptosis in non-small-cell lung cancer (NSCLC) cells is significantly dependent on EGFR status.61 In addition, dasatinib can block SFKs and downstream signaling components, such as FAK and p130Cas, and can suppress cell migration and invasion in sarcoma cell lines.62 Furthermore, an investigation with combined dasatinib plus JAK inhibitor I, also called pyridone 6 (P6), has provided a new strategy for inhibition of compensatory pathways in tumors.63

AZD0530, a potent dual-specific inhibitor of Src and ABL kinase, can prevent endocrine resistance when combined with other inhibitors. In breast cancer cells with mutated estrogen receptor, AZD0530 in combination with tamoxifen caused synergistic inhibition of Src.64 When AZD0530 was combined with an EGFR inhibitor, it prevented the emergence of an EGFR inhibitor-resistant state.65 SKI-606, a dual Src and ABL kinase inhibitor, has been shown to block BCR-ABL phosphorylation in chronic myelogenous leukemia cells, both in vitro and in vivo.66 However, SKI-606 substantially repressed the Src autophosphorylation site in HT29 and Colo205 tumors in an in vivo study of colorectal cancer.67 SKI-606 can suppress the cell growth, migration, and motility of colorectal cancer cells by blocking c-Src and its downstream signaling pathways.68

SFK inhibitors in clinical development

As SFKs are critical components in many intracellular processes that promote cancer progression, therapeutic strategies to inhibit SFKs are currently being developed. Dasatinib, approved for chronic myeloid leukemia (CML) and Philadelphia positive acute lymphoblastic leukemia, targets SFKs, BCR-ABL, c-kit, Eph, and PDGFR, as well as other kinases. AZD0530 is a potent, orally administered inhibitor of Src that has been shown in preclinical studies to prevent phosphorylation of downstream mediators of motility and invasion, paxillin and FAK, with inhibition of metastasis in vivo. SKI-606 (bosutinib) is an oral inhibitor of ABL and SFKs that inhibits Src-dependent tyrosine phosphorylation. Currently active clinical trials of these agents are listed in Table 1. It is important to note that SFK inhibitors such as dasatinib and bosutinib might have significant off-target effects on kinases other than SFKs, leading to differences in efficacy and toxicity.69,70

Preliminary results have been reported on phase I studies of SFK inhibitors in patients with cancer. Evans et al.71 reported on a phase I dose-escalation study of dasatinib in patients with advanced, treatment-refractory solid tumors. Maximum tolerated doses were established at 70 mg twice daily (every 12 h) for 5 consecutive days followed by 2 nontreatment days every week. Fourteen patients with Eastern Cooperative Oncology Group performance status <1 with gastrointestinal stromal tumors (n = 9) or other solid tumors (n = 5) were treated with one of three escalating dose levels: 35, 50, or 70 mg twice daily. Toxic effects included clinically insignificant grade 3 lymphopenia (two patients), grade 3 anorexia (one patient), and elevation of alkaline phosphatase to grade 3 (one patient). No dose-limiting toxicities were observed. A continuous, twice-daily schedule of 70 mg was well-tolerated and recommended for phase II development. Inhibition of phospho-Src seems to correlate with the plasma levels of dasatinib. Messersmith et al.72 presented a phase I study of bosutinib (SKI-606) in patients with a variety of solid tumors. Pharmacokinetic data indicate that this oral drug can be dosed once daily. Adverse effects were mainly gastrointestinal. Drug-related adverse events, of any grade, occurring in >25% of patients were nausea (67%), diarrhea (55%), anorexia (45%), vomiting (43%), and asthenia (41%). The only grade 3 drug-related adverse event occurring in >5% of patients was diarrhea (14%). Five patients had stable disease for more than 15 weeks (one with breast cancer, one with NSCLC, two with colorectal cancer). Three patients had stable disease for more than 24 weeks (one with breast cancer, one with lung cancer, one with pancreatic cancer [ongoing, more than 52 weeks]). The maximum tolerated dose was 400 mg daily, which will be evaluated in further clinical studies. In a phase I dose-finding study, AZD0530 was given to 81 patients, with doses ranging from 50 to 250 mg daily. In the first part of the study (part A), investigators defined the maximum tolerated dose, toxicity profile, and pharmacokinetics. In the second part (part B), the 50 mg, 125 mg, and 175 mg cohorts were expanded to characterize changes in phosphorylation of the Src substrates, paxillin and FAK, by evaluating intensity and localization of staining of these proteins by immunohistochemistry. In part A, dose-limiting toxic effects occurred in three patients at 250 mg (leucopenia, septic shock, renal failure, asthenia) and in two patients at 299 mg (febrile neutropenia, dyspnea). The 50, 125, and 175 mg doses were confirmed to be tolerable in part B. Pathology analysis revealed consistent modulation of phosphorylation and/or cellular localization of tumor paxillin and FAK consequent to AZD0530 therapy. There was a dose-response trend for reductions in paxillin phosphorylation. This study showed that AZD0530 is well tolerated at doses significant to inhibit Src phosphorylation.

Breast cancer

In breast cancer, Src promotes growth of tumor cells via interaction with human epidermal growth factor receptor 2 (HER2); Src activation is thought to be responsible for the high metastatic potential of HER2-overexpressing cancer.73 Src is important in the mediation of downstream effects of receptor tyrosine kinases, including HER2 and EGFR.74

c-Src transfection potentiates EGF-induced oncogenesis.75 Breast cancer cell lines that overexpress EGFR and Src have higher levels of phosphorylated Src, increased activation of MAPK duplication, and increased tumorigenicity compared with cell lines that do not overexpress EGFR or overexpress only Src.76 The use of an EGFR inhibitor combined with a Src inhibitor has shown significant suppression of migration and invasion in tamoxifen-resistant MCF-7 cells.77 In vivo studies have shown that the Src inhibitor SKI-606 successfully repressed breast tumor invasion, growth, and metastasis.78 The use of Src inhibitor combined with trastuzumab or lapatinib might halt metastasis.

Lung cancer

Increased levels of c-Src protein and/or kinase activity have been reported in 50–80% of patients with lung carcinoma.79 Preclinical studies have suggested that SFK inhibitors are active in models of lung cancer.61,80 One study is examining the combination of the EGFR inhibitor erlotinib in combination with dasatinib in patients with NSCLC previously refractory to chemotherapy.81 Another study is examining first-line dasatinib treatment in chemotherapy-naive patients with NSCLC and how this correlates with EGFR mutation status and phosphorylated Src, as measured by immunohistochemistry Based on preclinical studies that dasatinib and other SFK inhibitors can induce cell death in cell lines harboring activating EGFR mutations, researchers at Memorial Sloan-Kettering Cancer Center are evaluating dasatinib for patients who have acquired resistance to EGFR tyrosine kinase inhibitors. Finally, studies in small-cell lung cancer are also being conducted through the Cancer and Leukemia Group B cooperative network. These studies will probably have preliminary results within the next 1–2 years. The combination of cytotoxic chemotherapy, epidermal growth factor inhibitor, and a Src inhibitor could lead to additive effects in controlling tumor metastasis in patients with lung cancers that have an activated or mutated EGFR.

Prostate cancer

Src signaling promotes proliferation of prostate cancer cells in response to androgens and could contribute to androgen-independent prostate cancer via signaling from various factors, including growth factors and chemokines. Therefore, it seems likely that, along with antiandrogen deprivation therapy for prostate cancer, the addition of a Src inhibitor could have additive effects.82 Yu et al. conducted a phase II study of dasatinib in patients with hormone-refractory progressive prostate cancer, and assessed the response rate.83 In total, 46 men were enrolled. The initial starting dose was 100 mg twice daily, but after the first 25 patients were enrolled, the starting dose was changed to 70 mg twice daily for improved tolerability. The disease control rate for 15 patients was 67% (10 patients had stable disease). Of 27 patients who had bone scans at 12 weeks, 16 were stable and one was improved. Two of the five patients who had two or more bone scans at 24 weeks had stable disease. An improved prostate-specific antigen (PSA) doubling time was seen in 29 of 36 patients. One of 36 patients with two or more PSA measurements had a PSA response. Six patients experienced grade 1–2 pleural effusions and one patient had grade 3 pleural effusions at the 70 mg twice-daily dose. Ten patients experienced grade 1–2 effusions at the 100 mg twice-daily dose.

Colon cancer

In a panel of 28 human colon cancer cell lines, Wainberg et al.84 used microarray analysis to identify a three-gene set that was able to distinguish cell lines sensitive and resistant to dasatinib. These three genes (PTK-7, PLK-2, and PLK-3) had relatively high levels of expression in dasatinib-sensitive cell lines compared with dasatinib-insensitive cell lines. No mutations were found in the kinase domain of Src. These results suggest a role for dasatinib in the treatment of human colorectal cancer and identify potential markers of response.

Head and neck cancer

Koppikar et al.85 showed that squamous-cell carcinoma of the head and neck (HNSCC) expressing dominant active c-Src has increased growth and invasion compared with vector-transfected controls. Combined treatment with AZD0530 and gefitinib resulted in greater inhibition of HNSCC cell growth and invasion compared with either agent alone. The study showed that increased expression and activation of c-Src promotes HNSCC progression, and the combination of an EGFR and c-Src inhibitor might be a reasonable approach. EGFR seems to be upregulated in more than 90% of HNSCCs.86 High EGFR levels in patients with HNSCC have been associated with shorter relapse-free and overall survival times.87 Esteve et al.88 reported on a phase I trial of cetuximab and dasatinib in patients with advanced solid malignancies. In total, 11 patients were enrolled. Dasatinib 100–150 mg once daily by mouth, on a continuous schedule on days 1–21, was safely combined with cetuximab administered intravenously on a weekly schedule at a dose of 250 mg/m2 (after a loading dose of 400 mg/m2 on cycle 1, day 1) on days 1, 8, and 15. The predominant toxic effect was grade 1–2 headaches. Accrual continues on dose level 3 (dasatinib 200 mg), and pharmacokinetic and pharmacodynamic studies are planned.

Hematologic malignancies

Myeloid cells primarily express Lyn, Hck, and Fgr, all of which could all be implicated in SFK activity.89 The IL-3-induced upregulation of Lyn kinase activity is thought to be mediated by the 120 kDa common subunit of human IL-3 and granulocyte-macrophage colony-stimulating factor receptors.90 The activity of Lyn and Hck was increased in hematopoietic cells that overexpressed BCR-ABL.91

There is reported evidence that Lyn contributes to BCR-ABL cell motility through PI3K pathway signaling.92 Hu et al.93 used BCR-ABL1 retrovirus-transduced bone marrow from mice that lacked all three Src kinases and efficiently induced CML but not B-cell acute lymphoblastic leukemia (B-ALL) in recipients. The kinase inhibitor CGP76030 impaired in vitro proliferation of BCR-ABL-positive B-lymphoid cells and prolonged the survival of mice with B-ALL but not mice with CML. These results suggest that SFKs are required for BCR-ABL-positive B-lymphoblastic leukemia but not for CML.94

A phase I study of dasatinib treatment in patients with Philadelphia-chromosome-positive CML who did not respond or were intolerant to imatinib has been reported.95 Clinical activity with dasatinib was observed in all patients in this study except those with T315I mutations in BCR-ABL.96 Patients with Philadelphia-chromosome-positive ALL who were given dasatinib showed a high response rate, which suggests that Src could have a prominent role in this disease.93 Dasatinib has been approved by the FDA for patients with CML who have failed to respond or are intolerant to imatinib and for patients with Philadelphia-chromosome-positive ALL.

Src inhibitors on osteoclast function

Src has a precise and central role in osteoclast function by positively regulating osteoclasts and negatively regulating osteoblasts. Src activity is necessary for cytoskeletal organization in osteoclasts and bone resorbing activity.97 Src and other SFKs are involved in antiapoptotic signaling in osteoclasts induced by receptor activator for nuclear factor κB ligand (RANKL) and other tumor necrosis factor family members.98 Inhibition or disruption of Src decreases osteoblast proliferation but increases osteoblast differentiation and bone formation.99 During the bone resorption process, many growth factors are activated, such as transforming growth factor beta (TGF-β), insulin-like growth factors (IGF-I, II), FGFs, and PDGFs. These factors promote bone destruction in patients with metastatic bone disease. Studies in mice have shown that when Src is inhibited or disrupted, diminished bone metastases will occur.100

AZD0530, in a phase I study in patients with solid tumors, has been shown to decrease levels of bone resorption markers.101 One bone-targeted Src inhibitor that inhibits tyrosine kinase activity, APP22408, demonstrated antiosteoclast and antiresorptive activity both in vitro and in vivo.102 Another such inhibitor, AP23451, prevented osteolytic lesions in mice inoculated with a human breast cancer cell line.103

Future for Src inhibitors

As a classical oncogene and central mediator of signaling, SFKs have key roles in the maintenance of tumor cell biology. Despite a large body of information about the biology of SFKs, the translation of biological studies to clinical application might not be straightforward. One reason is the complexity of the SFK signaling pathways, including its ability to cooperate with multiple receptor tyrosine kinases as well as many downstream substrates. In addition, there is little evidence that Src is mutated or amplified to a great extent compared with other oncogenes, such as EGFR, HER2, and BCR-ABL.53 Choice of biomarkers for SFK activity and sensitivity to SFK inhibitors are also unclear. Bild et al.104 used gene expression signatures that reflected the activation status of several oncogenic pathways including Src. They used breast cancer cell lines to test the sensitivity of the cell lines to a Src inhibitor. In the future, these gene expression signatures may be used to predict sensitivity to therapeutic agents that target Src. Finally, SFK inhibitors such as dasatinib and bosutinib are not selective, and specific inhibitors of SFK alone can inhibit many other kinases with potential signaling effects on tumors cells and effects on normal tissues. Although the precise function of SFKs in human tumors has not yet been clearly defined, these basic biological studies for SFKs will finally provide the proper groundwork for cancer therapy in drug development and clinical studies.

Conclusions

For now, it will be important to determine if SFK inhibitors can induce tumor regressions or prolonged disease stability in patients with solid tumors. Combination strategies with key receptor tyrosine kinases are also important given the known cooperative roles of SFKs and receptor tyrosine kinases in tumor growth. Combining SFK inhibitors with other inhibitors of angiogenesis could also be important. Adjuvant therapy in solid tumors might include the long-term use of SFK inhibitors to prevent recurrence or metastasis. Continued work on understanding mechanisms of action of SFK inhibitors is important in being able to select or identify biomarkers that could predict optimal subsets of patients likely to benefit from these agents. Finally, creative use of these agents as antimetastatic treatments is warranted, and these studies will have to develop alternate end points for clinical trials compared to standard response rate assessments.

Review criteria

PubMed, Medline databases, the NIH clinical trials website, and Osprey network visualization system (version 1.2.0) via the BIO-GRID database were searched for articles published before 1 February 2009. Electronic early-release publications were also included. Only articles published in English were considered. The search terms used included “c-SRC” in association with the terms “reviews”, “Src inhibitors”, “structure of src” “dasatinib”, “SKI-606”, “AZD0530”, and “Src in cancer”. When possible, primary sources have been quoted.