Targeted therapy of orthotopic human lung cancer by combined vascular endothelial growth factor and epidermal growth factor receptor signaling blockade

The outcome for patients with lung cancer has not changed significantly for more than two decades. Several studies show that the overexpression of vascular endothelial growth factor (VEGF)/vascular permeability factor and epidermal growth factor (EGF) and their receptors correlates with the clinical outcome for lung cancer patients. However, clinical trials of agents that target either of these pathways alone have been disappointing. We hypothesize that targeting both the tumor and its vasculature by simultaneously blocking the VEGFR and EGFR pathways will improve the treatment of locoregional lung cancer. Human lung cancer specimens were first examined for the activation of VEGF receptor 2 (VEGFR2) and EGF receptor (EGFR) for tumor and tumor-associated endothelial cells, and both were found to be activated. The effects of ZD6474 (ZACTIMA), a small-molecule inhibitor of VEGFR2 and EGFR tyrosine kinases, were then studied in vitro using human lung cancer and microvascular endothelial cells. In vitro, ZD6474 inhibited EGFR, VEGFR2, mitogen-activated protein kinase and Akt phosphorylation, EGF- and VEGF-induced proliferation, and endothelial cell tube formation and also induced apoptosis. ZD6474 was further studied in vivo using an orthotopic mouse model of non-small cell lung cancer using NCI-H441 human lung adenocarcinoma cells. The inhibition of both VEGFR2 and EGFR signaling pathways by ZD6474 resulted in profound antiangiogenic, antivascular, and antitumor effects. These results provide a basis for the development of clinical strategies for the combination of selective protein tyrosine kinase inhibitors that block both EGFR and VEGFR signaling as part of the management of locally advanced lung cancer.     

The role of epidermal growth factor receptor in advanced non‐small cell lung carcinoma

Chemotherapy is the standard of care for patients with advanced non‐small cell lung cancer (NSCLC). Over the past 20 years, advances in chemotherapy have shown minimal incremental improvement in the survival outcomes of patients with advanced NSCLC. With the identification of molecular and genetic alterations in lung cancer, several new potential rationally designed therapeutic targets have emerged. One of these is the epidermal growth factor receptor (EGFR) and member of the ErbB family of receptor tyrosine kinases. Several inhibitors, both antibodies directed at the extra‐cellular portion of the receptor, and small molecule inhibitors directed at the tyrosine kinase domain of EGFR are in clinical development in lung cancer. This article will review the pre‐clinical rationale and the clinical studies of EGFR inhibitors alone and/or in combination with chemotherapy that have been performed to date in advanced NSCLC.     

The role of epidermal growth factor receptor in advanced non-small cell lung carcinoma

Chemotherapy is the standard of care for patients with advanced non-small cell lung cancer (NSCLC). Over the past 20 years, advances in chemotherapy have shown minimal incremental improvement in the survival outcomes of patients with advanced NSCLC. With the identification of molecular and genetic alterations in lung cancer, several new potential rationally designed therapeutic targets have emerged. One of these is the epidermal growth factor receptor (EGFR) and member of the ErbB family of receptor tyrosine kinases. Several inhibitors, both antibodies directed at the extra-cellular portion of the receptor, and small molecule inhibitors directed at the tyrosine kinase domain of EGFR are in clinical development in lung cancer. This article will review the pre-clinical rationale and the clinical studies of EGFR inhibitors alone and/or in combination with chemotherapy that have been performed to date in advanced NSCLC.     

Interaction between epidermal growth factor receptor- and cyclooxygenase 2-mediated pathways and its implications for the chemoprevention of head and neck cancer

Head and neck squamous cell carcinoma is a well-known model for chemoprevention studies because of its field cancerization effect, its multistep carcinogenesis process, and the easy accessibility of biopsies to target lesions. With new understandings of head and neck carcinogenesis and the development of molecular targeted therapy, chemoprevention trials for head and neck squamous cell carcinoma have been rapidly updated. Cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are gaining significant attention as potential chemopreventive agents. Both COX-2 and EGFR are involved in head and neck carcinogenesis. Targeting COX-2 and EGFR separately has shown promising antitumor activity. Recently, combinations of COX-2 and EGFR tyrosine kinase inhibitors have been reported to show synergistic/additive effects in preclinical studies. Because COX-2 and EGFR tyrosine kinase inhibitors are toxic as single agents in clinical trials, the combination of COX-2 and EGFR tyrosine kinase inhibitors used at lower doses seems more promising than monotherapy with either as a novel strategy in head and neck cancer chemoprevention.     

Inhibition of the epidermal growth factor receptor family of tyrosine kinases as an approach to cancer chemotherapy: progression from reversible to irreversible inhibitors.

The rationale to inhibit the epidermal growth factor receptor (EGFR) tyrosine kinase family as an approach to cancer chemotherapy has continued to grow stronger over the last 10 years. Both preclinical and clinical data strongly support the involvement of these receptors in the formation and progression of human cancers, as well as establish a high correlation in cancer patients between receptor/ ligand expression and poor prognosis. During the past 4 years, significant progress has been made in the area of EGFR tyrosine kinase inhibitors, and new structural classes have emerged that exhibit enormous improvements with regard to potency, specificity, and in vitro and in vivo activity. Very recently, further advancements in this field have been made whereby very specific, irreversible inhibitors of the EGFR family have been synthesized that provide unique pharmacological properties and exceptional efficacy. The in vivo performance of these modern kinase inhibitors has improved to the point where several compounds are either in clinical trials or very near to that point in their development. This review will briefly address the justification for targeting the EGFR family for cancer therapeutics, and then will highlight some of the more promising kinase inhibitors that are in development.     

Chasing targets for EGFR tyrosine kinase inhibitors in non-small-cell lung cancer: Asian perspectives

EGF receptor (EGFR) activation has an important role in various steps of carcinogenesis and progression of non-small-cell lung cancer (NSCLC), implying that EGFR is a potential target for cancer therapy. Therefore, targeted treatments aimed at EGFR have been developed, of which tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib have shown activity in NSCLC. The unexpected findings of a lack of association between expression of EGFR, the target and efficacy of EGFR TKIs and higher response in selective subgroups of patients were puzzling. Identification of somatic activating mutations in the EGFR tyrosine kinase domain solved the mystery and provided new insight. Several lines of study provided information on various molecular targets for EGFR TKI therapy. A target-based patient-selection strategy is expected to eventually lead to tailored therapy for lung cancer.     

The role of HER3 in gastric cancer

Gastric cancer is the second leading cause of cancer mortality in the world. HER family tyrosine kinases play a critical role in the development of gastric cancer. The HER family of receptor tyrosine kinases includes EGF receptor (EGFR), HER2, HER3, and HER4. Targeted drugs antineoplastic therapies such as EGFR tyrosine kinase inhibitors have application with confrontation of gastric cancer. However, less attention has been paid to the oncogenic functions of HER3 essepecially in the gastric cancer due to its lack of intrinsic kinase activity. Recent work, however, has placed the role of HER3 in gastric cancer in the spotlight as a key signaling hub in several contexts. First, HER3 overexpression may be associated with poor prognosis and unfavorable survival mediated by PI3K/AKT signaling pathway. Second, a large amount of direct evidence has emerged the benefit of anti-HER3 agents in combination with EGFR tyrosine kinase inhibitors as well as anti-HER2 agents in gastric cancer. Furthermore, we can further elucidate the relationship between HER3 and MET inhibitors in gastric cancer that the development of resistance to MET inhibitors may result from the overexpression of HER3. This review focuses on the current achievements of the relationship between HER3 and gastric cancer in vivo and in vitro, the development of HER3 molecule-targeted therapy, additionally, the challenge which we will meet in the future.     

The T790M "gatekeeper" mutation in EGFR mediates resistance to low concentrations of an irreversible EGFR inhibitor

Patients with non-small cell lung cancer (NSCLC) harboring activating mutations in the epidermal growth factor receptor (EGFR) kinase domain tend to respond well to the tyrosine kinase inhibitors, gefitinib and erlotinib. However, following clinical response, these patients typically relapse within a year of treatment. In many cases, resistance is caused by an acquired secondary EGFR kinase domain mutation, T790M. In vitro studies have shown that a new class of EGFR-irreversible inhibitors could overcome the resistance conferred by T790M. Clinical trials are under way to examine the efficacy of one of these inhibitors, HKI-272, in patients with NSCLC who initially responded to gefitinib/erlotinib and subsequently relapsed. To anticipate the possibility that patients who respond to irreversible inhibitors will develop secondary resistance to such inhibitors, as has been seen in other similar settings, we modeled acquired resistance to the dual EGFR/HER2-irreversible tyrosine kinase inhibitor HKI-272 in a NSCLC cell culture model. We found that HKI-272-resistant clones fall into two biochemical groups based on the retention of EGFR phosphorylation in the presence of the drug. Cells that retain phosphorylated EGFR have acquired the secondary mutation T790M. Moreover, HKI-272 can overcome T790M resistance only at suprapharmacologic concentrations. We further model mutations at EGFR C797 as a mechanism of resistance to irreversible EGFR inhibitors and show that although these mutants are resistant to the irreversible inhibitor, they retain erlotinib sensitivity. Our findings suggest that HKI-272 treatment at maximally tolerated dosing may lead to the emergence of T790M-mediated resistance, whereas treatment with a more potent irreversible inhibitor could yield a resistance mutation at EGFR C797.     

Diagnostic and therapeutic biomarkers for lung cancer patients

Identification of sensitive biomarkers predictive of diagnosis, prognosis and drug sensitivity could have a clinically significant impact on non-small cell lung cancer (NSCLC) treatment strategies. Recently, molecular-targeted therapies have been developed for NSCLC treatment. NSCLC patients with epidermal growth factor receptor (EGFR) gene mutations have shown a dramatic response to EGFR tyrosine kinase inhibitors (EGFR-TKI) such as gefitinib and erlotinib. In addition, target therapies against echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion protein present in approximately 5% of the Japanese patients with adenocarcinomas are currently under development. In this paper, we reviewed diagnostic and therapeutic biomarkers for lung cancer patients.     

放疗联合表皮生长因子受体酪氨酸激酶抑制剂药物治疗晚期非小细胞肺癌的研究进展

靶向药物表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)通过竞争性阻断表皮生长因子受体(EGFR)转导的自身磷酸化、调控细胞周期、阻碍肿瘤细胞DNA损伤修复以及抑制新生血管形成等,起到放疗增敏的效果,同时不增加毒副反应的发生。目前放疗联合EGFR-TKI药物在治疗晚期非小细胞肺癌(NSCLC)及转移灶的临床应用中已显示出一定的疗效优势,有望成为EGFR突变的NSCLC优势人群另一项重要的治疗措施。     

Cigarette smoke induces aberrant EGF receptor activation that mediates lung cancer development and resistance to tyrosine kinase inhibitors

The EGF receptor (EGFR) and its downstream signaling are implicated in lung cancer development. Therefore, much effort was spent in developing specific tyrosine kinase inhibitors (TKI) that bind to the EGFR ATP-pocket, blocking EGFR phosphorylation/signaling. Clinical use of TKIs is effective in a subset of lung cancers with mutations in the EGFR kinase domain, rendering the receptor highly susceptible to TKIs. However, these benefits are limited, and emergence of additional EGFR mutations usually results in TKI resistance and disease progression. Previously, we showed one mechanism linking cigarette smoke to EGFR-driven lung cancer. Specifically, exposure of lung epithelial cells to cigarette smoke-induced oxidative stress stimulates aberrant EGFR phosphorylation/activation with impaired receptor ubiquitination/degradation. The abnormal stabilization of the activated receptor leads to uncontrolled cell growth and tumorigenesis. Here, we describe for the first time a novel posttranslational mechanism of EGFR resistance to TKIs. Exposure of airway epithelial cells to cigarette smoke causes aberrant phosphorylation/activation of EGFR, resulting in a conformation that is different from that induced by the ligand EGF. Unlike EGF-activated EGFR, cigarette smoke-activated EGFR binds c-Src and caveolin-1 and does not undergo canonical dimerization. Importantly, the cigarette smoke-activated EGFR is not inhibited by TKIs (AG1478; erlotinib; gefitinib); in fact, the cigarette smoke exposure induces TKI-resistance even in the TKI-sensitive EGFR mutants. Our findings show that cigarette smoke exposure stimulates not only aberrant EGFR phosphorylation impairing receptor degradation, but also induces a different EGFR conformation and signaling that are resistant to TKIs. Together, these findings offer new insights into cigarette smoke-induced lung cancer development and TKI resistance.     

The EGFR T790M mutation in acquired resistance to an irreversible second-generation EGFR inhibitor

Molecular target therapies using first-generation, reversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as gefitinib or erlotinib, have been shown to be effective for patients with non-small cell lung cancer (NSCLC) who harbor activating mutations in EGFR. However, these patients eventually develop resistance to the reversible TKIs, and this has led to the development of second-generation, irreversible EGFR inhibitors. Currently, the mechanism of acquired resistance to irreversible EGFR inhibitors is not clear. Using an in vitro cell culture system, we modeled the acquired resistance to first-line treatment with second-generation EGFR-TKIs using an EGFR-mutant NSCLC cell line. Here, we report a mechanism of resistance involving T790M secondary mutation as well as a corresponding clinical case. The results of these findings suggest that inhibition of EGFR by currently available second-generation EGFR-TKIs may not be sufficient to physiologically prevent the emergence of cells that are still dependent on EGFR signaling. This finding bears important implications on the limitations of currently available second-generation EGFR-TKIs.     

E7080 suppresses hematogenous multiple organ metastases of lung cancer cells with nonmutated epidermal growth factor receptor

While epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors improve the prognosis of patients with EGFR mutant lung cancer, the prognosis of patients with nonmutant EGFR lung cancer, especially those with metastases, is still extremely poor. We have assessed the therapeutic efficacy of E7080, an orally available inhibitor of multiple tyrosine kinases including VEGF receptor 2 (VEGFR-2) and VEGFR-3, in experimental multiple organ metastasis of lung cancer cell lines without EGFR mutations. E7080 markedly inhibited the in vitro proliferation of VEGF-stimulated microvascular endothelial cells. Intravenous inoculation into natural killer cell-depleted severe combined immunodeficient mice of the small cell lung cancer cell lines H1048 (producing low amounts of VEGF) and SBC-5 (producing intermediate amounts of VEGF) resulted in hematogenous metastases into multiple organs, including the liver, lungs, kidneys, and bones, whereas intravenous inoculation of PC14PE6, a non-small cell lung cancer cell line producing high amounts of VEGF, resulted in lung metastases followed by massive pleural effusion. Daily treatment with E7080 started after the establishment of micrometastases significantly reduced the number of large (>2 mm) metastatic nodules and the amount of pleural effusion, and prolonged mouse survival. Histologically, E7080 treatment reduced the numbers of endothelial and lymph endothelial cells and proliferating tumor cells and increased the number of apoptotic cells in metastatic nodules. These results suggest that E7080 has antiangiogenic and antilymphangiogenic activity and may be of potential therapeutic value in patients with nonmutant EGFR lung cancer and multiple organ metastases.     

非小细胞肺癌的EGFR表达与EGFR基因突变的比较

目的探讨非小细胞肺癌（NSCLC）表皮生长因子受体（EGFR）免疫组化表达与EGFR基因突变的关系及其临床意义。方法对130例手术切除的非小细胞肺癌组织应用免疫组化方法检测EGFR表达,同时应用基因测序方法检测EGFR基因突变,研究EGFR表达与EGFR基因突变的关系。结果130例NSCLC中有38例EGFR基因突变,突变率为29．2％。EGFR阳性率为67．7％。EGFR表达与基因突变无关（P〉0．05）,结论EGFR免疫组化表达与EGFR基因突变无关,不能作为筛选EGFR酪氨酸激酶抑制剂（TKI）治疗NSCLC的指标。     

Dual mechanisms of action of the 5-benzylidene-hydantoin UPR1024 on lung cancer cell lines

In this study, we examined the mechanism of action of the novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor 5-benzylidene-hydantoin UPR1024, whose structure was designed to interact at the ATP-binding site of EGFR. The compound had antiproliferative and proapoptotic effects when tested on the non-small cell lung cancer cell line A549. The growth inhibitory effect was associated with an accumulation of the cells in the S phase of the cell cycle. Moreover, UPR1024 induced significant level of DNA strand breaks associated with increased expression of p53 and p21(WAF1) proteins, suggesting an additive mechanism of action. The presence of wild-type p53 improved the drug efficacy, although the effect was also detectable in p53 null cells. We also noted apoptotic cell death after treatment with UPR1024 at concentrations above 10 mumol/L for >24 h, with involvement of both the extrinsic and intrinsic pathways. The present data show that UPR1024 may be considered a combi-molecule capable of both blocking EGFR tyrosine kinase activity and inducing genomic DNA damage. UPR1024 or its derivatives might serve as a basis for development of drugs for the treatment of lung cancer in patients resistant to classic tyrosine kinase inhibitors.     

Dual targeting of EGFR can overcome a major drug resistance mutation in mouse models of EGFR mutant lung cancer

EGFR is a major anticancer drug target in human epithelial tumors. One effective class of agents is the tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. These drugs induce dramatic responses in individuals with lung adenocarcinomas characterized by mutations in exons encoding the EGFR tyrosine kinase domain, but disease progression invariably occurs. A major reason for such acquired resistance is the outgrowth of tumor cells with additional TKI-resistant EGFR mutations. Here we used relevant transgenic mouse lung tumor models to evaluate strategies to overcome the most common EGFR TKI resistance mutation, T790M. We treated mice bearing tumors harboring EGFR mutations with a variety of anticancer agents, including a new irreversible EGFR TKI that is under development (BIBW-2992) and the EGFR-specific antibody cetuximab. Surprisingly, we found that only the combination of both agents together induced dramatic shrinkage of erlotinib-resistant tumors harboring the T790M mutation, because together they efficiently depleted both phosphorylated and total EGFR. We suggest that these studies have immediate therapeutic implications for lung cancer patients, as dual targeting with cetuximab and a second-generation EGFR TKI may be an effective strategy to overcome T790M-mediated drug resistance. Moreover, this approach could serve as an important model for targeting other receptor tyrosine kinases activated in human cancers.     

Combine therapy of gefitinib and fulvestrant enhances antitumor effects on NSCLC cell lines with acquired resistance to gefitinib

Gefitinib, an EGFR receptor tyrosine kinase inhibitor, is approved for clinical use in the treatment of non-small cell lung cancer (NSCLC), but the emergence of mutations resistant to these inhibitors, such as T790M, has become a clinical problem. According to statistics, female patients, the presence of adenocarcinoma or non-smokers experienced a higher response rate. This may be involved in interaction between the estrogen receptor (ER) and the epidermal growth factor receptor (EGFR). To test whether inhibition of the ER signaling pathway affects the antitumor effect of gefitinib, gefitinib and an ER antagonist, fulvestrant, were administered to NSCLC cell lines with acquired resistance to gefitinib. Compared with treatment of either fulvestrant or gefitinib alone, drug combination obviously decreased proliferation of H1976, H1650 and PC-9 cells coming from adenocarcinoma. Rapid activations of EGFR pathway by E2β were observed in H1975 cells with T790M mutation. Additionally, EGFR and ERs expression were down-regulated respectively in response to estrogen and EGF but up-regulated in response to fulvestrant and gefitinib in vitro. These results suggest that there is a functional cross-signaling between the EGFR/ER pathways in NSCLC with acquired resistance to gefitinib, possibly providing rationale for combining gefitinib with anti-estrogen therapy for advanced NSCLC treatment.     

Strategies toward the design of novel and selective protein tyrosine kinase inhibitors.

Protein tyrosine kinases play a fundamental role in signal transduction pathways. Deregulated tyrosine kinase activity has been observed in many proliferative diseases (e.g., cancer, psoriasis, restenosis, etc.). Tyrosine kinases are, therefore, attractive targets for the design of new therapeutic agents against cancer. We have built up a pharmacophore model of the ATP-binding site of the epidermal growth factor receptor (EGFR) kinase and used it for the rational design of kinase inhibitors. Several examples of the successful use of this model are presented in this review. Amongst these, 4-substituted-pyrrolo[2,3-d]pyrimidines, a new class of highly potent and selective inhibitors of the EGFR kinase, have been identified and further optimized. The most active derivatives inhibited the EGFR tyrosine kinase with IC50 values between 1 and 5 nM. In EGF-dependent cellular systems, tyrosine phosphorylation, as well as c-fos mRNA expression, was inhibited with similar IC50 values. Further successful application of this pharmacophore model led to the identification and optimization of phenylamino-pyrazolo[4,3-d]pyrimidines and substituted isoflavones and quinolones, other classes of potent, selective, and ATP competitive EGFR kinase inhibitors with IC50 values in the low nanomolar range. Structure-activity relationships of both classes are discussed.     

HER kinase activation confers resistance to MET tyrosine kinase inhibition in MET oncogene-addicted gastric cancer cells

Tumor cells with genomic amplification of MET display constitutive activation of the MET tyrosine kinase, which renders them highly sensitive to MET inhibition. Several MET inhibitors have recently entered clinical trials; however, as with other molecularly targeted agents, resistance is likely to develop. Therefore, elucidating possible mechanisms of resistance is of clinical interest. We hypothesized that collateral growth factor receptor pathway activation can overcome the effects of MET inhibition in MET-amplified cancer cells by reactivating key survival pathways. Treatment of MET-amplified GTL-16 and MKN-45 gastric cancer cells with the highly selective MET inhibitor PHA-665752 abrogated MEK/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT signaling, resulting in cyclin D1 loss and G(1) arrest. PHA-665752 also inhibited baseline phosphorylation of epidermal growth factor receptor (EGFR) and HER-3, which are transactivated via MET-driven receptor cross-talk in these cells. However, MET-independent HER kinase activation using EGF (which binds to and activates EGFR) or heregulin-beta1 (which binds to and activates HER-3) was able to overcome the growth-inhibitory effects of MET inhibition by restimulating MEK/MAPK and/or PI3K/AKT signaling, suggesting a possible escape mechanism. Importantly, dual inhibition of MET and HER kinase signaling using PHA-665752 in combination with the EGFR inhibitor gefitinib or in combination with inhibitors of MEK and AKT prevented the above rescue effects. Our results illustrate that highly targeted MET tyrosine kinase inhibition leaves MET oncogene-"addicted" cancer cells vulnerable to HER kinase-mediated reactivation of the MEK/MAPK and PI3K/AKT pathways, providing a rationale for combined inhibition of MET and HER kinase signaling in MET-amplified tumors that coexpress EGFR and/or HER-3.