Activated Src and Ras induce gefitinib resistance by activation of signaling pathways downstream of epidermal growth factor receptor in human gallbladder adenocarcinoma cells

Purpose: Although gefitinib, a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, has been demonstrated to exhibit its antitumor activity by the blockade of EGF receptor, the role of signaling pathways downstream of EGFR in gefitinib sensitivity remains unknown. In this study, we investigated the mechanistic role of Src and Ras, major oncogene products implicated in the pathogenesis of many human cancers in gefitinib sensitivity. Methods: Using parental and v-src- or c-H-ras-transfected HAG-1 human gallbladder adenocarcinoma cell lines, effects of gefitinib on cytotoxicity, cell cycle purtubation and apoptosis, and tyrosine phosphorylation of EGFR, Akt, and Erk were determined by WST-1 assay, flow cytometry, and Western blots, respectively. Results: Activated Ras and Src conferred a strong resistance to gefitinib by nearly 30-fold and 200-fold, respectively. Gefitinib induced accumulation of cells in the G0/G1 phase of the cell cycle at 24-h, with progressive expansion of apoptotic cell population in parental HAG-1 cells, but these effects were completely abolished in v-src- or c-H-ras-transfected cell line. Upon gefitinib treatment, EGFR activation and subsequent downstream activation through Erk and Akt were significantly inhibited in HAG-1 cells. By contrast, gefinitib failed to inhibit the activation of both Akt and Erk in v-src-transfected cells and Erk, but not Akt in c-H-ras-transfected cells, despite the blockade of EGFR activation in these respective cell lines. Treatment of v-src-transfected cells with herbimycin A, a Src tyrosine kinase inhibitor, partially reversed the gefitinib resistance, with concomitant inhibition of Akt and Erk. Conclusion: Our results suggest that activated Ras and Src could induce gefitinib resistance by activating either or both of Akt and Erk signaling pathways, thus providing a strategic rationale for assessment of these specific signaling molecules downstream of EGFR to customize treatment.Baoli Qin and Hiroshi Ariyama contributed equally to this work.     

Epidermal growth factor receptor as a potential therapeutic target in triple-negative breast cancer

BackgroundNo proven targeted therapy is currently available for the treatment of triple-negative breast cancer (TNBC). Epidermal growth factor receptor (EGFR) is frequently overexpressed in TNBC. We studied the activity of EGFR antagonists alone, and in combination with chemotherapy, in TNBC cell lines.Materials and methodsEGFR and phosphorylated EGFR were measured by enzyme-linked immunosorbent assay. Sensitivity to EGFR inhibitors alone and in combination with chemotherapy was assessed. Effects of gefitinib on EGFR signalling and cell cycle were also examined.ResultsEGFR was overexpressed in the TNBC compared with the human epidermal growth factor receptor 2 (HER-2)-positive cell lines. Phosphorylation of EGFR was detected in the TNBC cells in response to epidermal growth factor stimulation and was blocked by gefitinib treatment. However, the TNBC cell lines were less sensitive to EGFR inhibition than the HER-2-positive cell lines. Response to gefitinib was associated with reduced phosphorylation of both mitogen activated protein kinase (MAPK) and Akt and induction of G₁ arrest. Gefitinib enhanced response to both carboplatin and docetaxel in the TNBC cells, and the triple combination of gefitinib, carboplatin and docetaxel was synergistic.ConclusionsAlthough the TNBC cells are less sensitive to EGFR inhibition than the HER-2-positive cell lines, gefitinib enhanced response to chemotherapy. Gefitinib combined with carboplatin and docetaxel warrants further investigation in TNBC.     

Uncoupling between epidermal growth factor receptor and downstream signals defines resistance to the antiproliferative effect of Gefitinib in bladder cancer cells

Activation of the epidermal growth factor receptor (EGFR) and downstream signaling pathways, such as phosphatidylinositol-3 kinase/Akt and Ras/mitogen-activated protein kinase (MAPK), have been implicated in causing resistance to EGFR-targeted therapy in solid tumors, including the urogenital tumors. To investigate the mechanism of resistance to EGFR inhibition in bladder cancer, we compared EGFR tyrosine kinase inhibitor (Gefitinib, Iressa, ZD1839) with respect to its inhibitory effects on three kinases situated downstream of EGFR: MAPK, Akt, and glycogen synthase kinase-3beta (GSK-3beta). We found that the resistance to the antiproliferative effects of gefitinib, in vitro as well as in vivo in nude mice models, was associated with uncoupling between EGFR and MAPK inhibition, and that GSK-3beta activation and degradation of its target cyclin D1 were indicators of a high cell sensitivity to gefitinib. Further analysis of one phenotypic sensitive (253J B-V) and resistant (UM-UC13) cell lines revealed that platelet-derived growth factor receptor-beta (PDGFRbeta) activation was responsible for short circuiting the EGFR/MAPK pathway for mitogenic stimuli. However, invasion as well as actin dynamics were efficiently reduced by EGFR inhibition in UM-UC13. Chemical disruption of signaling pathways or of PDGFR kinase activity significantly reduced the inactive pool of cellular GSK-3beta in UM-UC13 cells. In conclusion, our data show that the uncoupling of EGFR with mitogenic pathways can cause resistance to EGFR inhibition in bladder cancer. Although this uncoupling may arise through different mechanisms, we suggest that the resistance of bladder cancer cells to EGFR blockade can be predicted early in the course of treatment by measuring the activation of GSK-3beta and of nuclear cyclin D1.     

Evaluation of the therapeutic potential of the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib in preclinical models of bladder cancer.

The epidermal growth factor receptor (EGFR) is associated with aggressive phenotypes and is an independent predictor of stage progression and mortality in bladder cancer. Gefitinib ('Iressa,' ZD1839) is an orally active EGFR-tyrosine kinase inhibitor. The objective of this study was to evaluate the in vitro and in vivo effects of gefitinib in the EGFR-expressing human bladder cancer cell lines 253J B-V, RT-112, and T24. EGFR expression was 3- and 2-fold higher in 253J B-V and RT-112, respectively, compared with T24 cells. Ten microm gefitinib inhibited EGFR, p42/44 extracellular signal-regulated kinase (ERK), and Akt/protein kinase B phosphorylation in all three of the cell lines. Inhibition of ERK by gefitinib was significantly greater in 253J B-V compared with RT-112 and T24 cells (9:2:1 in 253J B-V:RT-112:T24), whereas inhibition of Akt phosphorylation was less in 253J B-V compared with RT-112 and T24 cells (1:9:30 in 253J B-V:RT-112:T24). When cultured in serum-free medium supplemented with epidermal growth factor, 10 microm gefitinib inhibited DNA synthesis in T24 and RT-112 cells, whereas 1 microm gefitinib was sufficient to inhibit DNA synthesis in 253J B-V cells. Similarly, in the presence of serum, 10 microm gefitinib induced a significant reduction in S-phase and viable cell number in T24 and RT-112 cells, whereas 1-10 microm gefitinib caused a dose-dependent effect on these phenotypes in 253J B-V cells. Gefitinib significantly enhanced the ability of ionizing radiation to reduce colony forming ability in 253J B-V and RT-112 cells. In nude mice, a daily oral dose of 150 mg/kg gefitinib induced regression of tumors produced by 253J B-V cells growing at s.c. sites and suppression of tumors produced by these cells at orthotopic sites but had no effect on tumors produced by RT-112 cells growing at s.c. sites. The data indicates that gefitinib has potential therapeutic value, alone or in combination with ionizing radiation, in a subset of EGFR-expressing bladder cancers. However, there is a differential response to gefitinib in these EGFR-expressing bladder cancer cell lines. Although gefitinib can inhibit phosphorylation of EGFR, ERK, and Akt, and inhibit growth of bladder cancer cells in vitro, it does not necessarily inhibit growth of bladder cancer cells in vivo. It is likely that optimized therapy approaches will require an accurate "molecular" diagnosis allowing effective, selective, tailored therapeutic strategies to be designed.     

Gefitinib inhibits MUC5AC synthesis in mucin-secreting non-small cell lung cancer cells

Gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is an active agent in non-small cell lung cancer, and rapidly relieves bronchorrhea in patients with bronchioloalveolar carcinoma before the improvement of radiological findings. In addition, epidermal growth factor regulates mucin secretion in normal airway goblet cells. The present study was designed to clarify whether gefitinib modifies mucin production in lung cancer cell lines apart from its anti-proliferative effects, using A549 adenocarcinoma and NCI-H292 mucoepidermoid carcinoma cells expressing EGFR and MUC5AC mRNA. Mucin synthesis was measured by RT-PCR and ELISA, and MAPK and Akt, the downstream targets of EGFR, were examined by Western blotting assay. The clinically-achievable concentration of 1muM gefitinib inhibited the growth of both cells by only 10%, but gefitinib suppressed MUC5AC mRNA levels subsequent to a decrease in intracellular and secreted MUC5AC protein. Gefitinib also inhibited the phosphorylation of MAPK and Akt, and the selective inhibitors PD98059 and LY294002 also suppressed MUC5AC protein synthesis. These findings suggest that gefitinib may inhibits MUC5AC synthesis, at least in part, through MAPK and Akt signaling pathways. Thus, gefitinib inhibits mucin production, which is encouraging for trials involving its use against bronchorrhea in patients with lung cancer.     

Better survival with EGFR exon 19 than exon 21 mutations in gefitinib-treated non-small cell lung cancer patients is due to differential inhibition of downstream signals

Somatic mutations in the epidermal growth factor receptor (EGFR) kinase domain are associated with sensitivity to tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC). Our clinical data showed NSCLC patients with exon 19 deletions survived longer following gefitinib treatment than those with exon 21 point mutations. We aimed to investigate whether these two mutations produced differences in phosphorylation of EGFR and downstream signals. Two stable cell lines expressing these mutations were obtained by transfection. Inhibition of phosphorylation of EGFR, Akt, and Erk by gefitinib was detected using Western blotting, and cell inhibition tests were conducted to evaluate the bio-behavior. Gefitinib inhibited the phosphorylation of EGFR, Akt, and Erk to a greater degree in exon 19 deletion cells than in L858R cells. Gefitinib produced G1 arrest in more of the cells with exon 19 deletion than with L858R. This might be attributable to patient selection in TKIs therapy.     

Chemoresistant tumor cell lines display altered epidermal growth factor receptor and HER3 signaling and enhanced sensitivity to gefitinib

Deregulated signaling through the epidermal growth factor receptor (EGFR) is involved in chemoresistance. To identify the molecular determinants of sensitivity to the EGFR inhibitor gefitinib (Iressa, ZD1839) in chemoresistance, we compared the response of matched chemosensitive and chemoresistant glioma and ovarian cancer cell lines. We found that chemoresistant cell lines were 2- to 3-fold more sensitive to gefitinib growth-inhibitory effects, because of decreased proliferation rather than survival. Sensitivity to gefitinib correlated with overexpression and constitutive phosphorylation of HER2 and HER3, but not EGFR, altered HER ligand expression, and enhanced activation of EGF-triggered EGFR pathway. No activating mutations were found in EGFR. Gefitinib fully inhibited EGF-induced and constitutive Akt activation only in chemoresistant cells. In parallel, gefitinib downregulated constitutively phosphorylated HER2 and HER3, and activated GSK3beta with a concomitant degradation of cyclin D1. Ectopically overexpressed HER2 on its own was insufficient to sensitize chemonaive cells to gefitinib. pHER3 coimmunoprecipitated with p85-PI3K in chemoresistant cells and gefitinib dissociated these complexes. siRNA-mediated inhibition of HER3 decreased constitutive activation of Akt and sensitivity to gefitinib in chemoresistant cells. Our study indicates that in chemoresistant cells gefitinib inhibits both an enhanced EGF-triggered pathway and a constitutive HER3-mediated Akt activation, indicating that inhibition of HER3 together with that of EGFR could be relevant in chemorefractory tumors. Furthermore, in combination experiments gefitinib enhanced the effects of coadministered drugs more in chemoresistant than chemosensitive ovarian cancer cells. Combined treatment might be therapeutically beneficial in chemoresistant tumors from ovary and likely from other tissues.     

Transforming growth factor alpha expression drives constitutive epidermal growth factor receptor pathway activation and sensitivity to gefitinib (Iressa) in human pancreatic cancer cell lines

The epidermal growth factor receptor (EGFR) is considered an important therapeutic target in pancreatic cancer, but it is currently impossible to identify those patients who are most likely to benefit from EGFR-directed therapy. We examined the biological effects of the EGFR tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) in a panel of nine human pancreatic cancer cell lines. The drug strongly inhibited DNA synthesis and induced low levels of apoptosis at clinically relevant concentrations in a subset of three of the lines (L3.6pl, BxPC3, and Cfpac1). Sensitivity to gefitinib correlated directly with ligand [transforming growth factor-alpha (TGF-alpha)] expression (r(2) = 0.71, P = 0.004) but not with surface EGFR expression. The gefitinib-sensitive cells displayed constitutive baseline EGFR phosphorylation, whereas the gefitinib-resistant cells did not. Exposure to gefitinib or a small interfering RNA construct specific for TGF-alpha reversed the constitutive EGFR phosphorylation and downstream target [extracellular signal-regulated kinases (ERK), AKT] phosphorylation in the gefitinib-sensitive cells but had no effects on ERK or AKT phosphorylation in gefitinib-resistant cells. Baseline EGFR phosphorylation was lower in a subclone of L3.6pl selected for low TGF-alpha expression, and these cells were also resistant to gefitinib-mediated growth inhibition. Gefitinib blocked the growth of tumor xenografts derived from L3.6pl cells but had no effect on the growth of tumors derived from EGFR-independent MiaPaCa-2 cells. Together, our data show that TGF-alpha expression identifies a subset of human pancreatic cancer cells that is dependent on EGFR signaling in vitro and in vivo. Quantification of TGF-alpha expression may therefore represent an effective means of identifying EGFR-responsive primary tumors.     

Additive antitumour effect of the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib (Iressa, ZD1839) and the antioestrogen fulvestrant (Faslodex, ICI 182,780) in breast cancer cells

A high expression level of epidermal growth factor receptor (EGFR)/HER1 has been suggested to lead to a shorter survival time and resistance to endocrine therapy in patients with breast cancer. To test the hypothesis that inhibition of the EGFR signalling pathway affects the antitumour effect of endocrine therapy, an EGFR tyrosine kinase inhibitor (EGFR-TKI), gefitinib, and an oestrogen receptor (ER) antagonist, fulvestrant, were administered to human breast cancer cells. A total of five human breast cancer cell lines were used. The effects of single or combined treatments with gefitinib and/or fulvestrant on cell growth, cell cycle progression and apoptosis were analysed. Changes in the expression levels of cyclin-dependent kinase inhibitors, p21 and p27, an antiapoptotic factor, Bcl-2, and a proapoptotic factor, Bax, were also investigated. All cell lines tested were sensitive to gefitinib (50% growth inhibitory concentration, 10-28.5 microM). Breast cancer cell lines with a high expression level of HER1 or HER2 were more sensitive to gefitinib than the others. Gefitinib induced a significant G1-S blockade in ER-positive KPL-3C cells. Gefitinib induced significant apoptosis in HER1-overexpressing MDA-MB-231 cells. Gefitinib additively increased the antitumour effect of fulvestrant in all three ER-positive cell lines in a medium supplemented with 17beta-oestradiol. The combined treatment promoted cell cycle retardation in KPL-3C cells, which is associated with an upregulation of p21 by fulvestrant and gefitinib, respectively. Apoptosis was associated with downregulation of Bcl-2 by gefitinib in MDA-MB-231 cells. These results suggest an additive interaction between the EGFR-TKI gefitinib and the antioestrogen fulvestrant in ER-positive breast cancer cells.     

Gefitinib ('Iressa', ZD1839), a selective epidermal growth factor receptor tyrosine kinase inhibitor, inhibits pancreatic cancer cell growth, invasion, and colony formation

Pancreatic cancer is a devastating malignancy, characterized by low responsiveness to conventional chemotherapies. Gefitinib, an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) has shown clinical activity against EGFR-expressing tumors. Since pancreatic cancers frequently overexpress EGFR (ErbB-1) and its ligands, our aim was to investigate the potential role of gefitinib in this disease. The GI50 of gefitinib as well as the effects of gefitinib on growth factor actions in pancreatic cancer cell lines were analyzed using MTT assays. FACS analysis using Annexin and propidium iodide (PI) staining were performed to study cell cycle, apoptosis and cell death. Western blot analysis was carried out to investigate expression levels of the 4 members of the ErbB family of receptors in pancreatic cancer cell lines, as well as MAP kinase and EGFR phosphorylation. Soft agar assays were used to measure colony formations. Invasiveness of cancer cells was analyzed using Matrigel-coated filters. gefitinib inhibited cell proliferation of pancreatic cancer cell lines with GI50 concentrations ranging from 2.5 to over 10 micro M. Gefitinib completely inhibited EGF-induced cell proliferation, but did not significantly influence insulin-like growth factor (IGF)-induced mitogenesis. Gefitinib also completely abolished EGF-induced phosphorylation of EGFR and MAP kinase. Furthermore, gefitinib inhibited basal and EGF-induced anchorage-independent cell growth and invasion. Our data demonstrate that gefitinib inhibits pancreatic cancer cell growth through EGFR-dependent pathways. Gefitinib also inhibits anchorage-independent growth and invasiveness, suggesting that gefitinib may offer a new approach for the treatment of pancreatic cancer.     

Gastrin-releasing peptide receptor mediates activation of the epidermal growth factor receptor in lung cancer cells

Gastrin-releasing peptide receptor (GRPR) and the epidermal growth factor receptor (EGFR) are expressed in several cancers including non-small cell lung cancer (NSCLC). Here we demonstrate the activation of EGFR by the GRPR ligand, gastrin-releasing peptide (GRP), in NSCLC cells. GRP induced rapid activation of p44/42 MAPK in lung cancer cells through EGFR. GRP-mediated activation of MAPK in NSCLC cells was abrogated by pretreatment with the anti-EGFR-neutralizing antibody, C225. Pretreatment of NSCLC cells with neutralizing antibodies to the EGFR ligands, TGF-A or HB-EGF, also decreased GRP-mediated MAPK activation. On matrix metalloproteinase (MMP) inhibition, GRP failed to activate MAPK in NSCLC cells. EGF and GRP both stimulated NSCLC proliferation, and inhibition of either EGFR or GRPR resulted in cell death. Combining a GRPR antagonist with the EGFR tyrosine kinase inhibitor, gefitinib, resulted in additive cytotoxic effects. Additive effects were seen at gefitinib concentrations from 1 to 18 microM, encompassing the ID50 values of both gefitinib-sensitive and gefitinib-resistant NSCLC cell lines. Because a major effect of GRPR appears to be promoting the release of EGFR ligand, this study suggests that a greater inhibition of cell proliferation may occur by abrogating EGFR ligand release in consort with inhibition of EGFR.     

PLC and PI3K Pathways are Important in the Inhibition of EGF-lnduced Cell Migration by Gefitinib (‘lressa’, ZD1839)

BACKGROUND: Expression of epidermal growth factor receptor (EGFR) by human breast cancer tissues is associated with poor clinical response. The EGFR tyrosine kinase inhibitor (EGFR-TKI), gefitinib ('Iressa', ZD1839), is a leading example of a molecular targeted agent, and has an anti-proliferative effect on various cancer cells. But the details of the anti-cancer effect and mechanism have not been elucidated. We studied the anti-cancer effect of gefitinib in breast cancer cell lines and the intracellular pathway downstream of EGFR associated with cell migration. METHODS: In this study, we analysed the anti-proliferative and anti-migratory effect of gefitinib in EGFR (+) breast cancer cell lines by WST-1 analysis and chemotaxis chamber analysis. We analyzed several intracellular phosphorylated pathways which are activated by mitogen activated kinases (extracellular signal-regulated protein kinase 1 and 2: MEK), phosphatidylinositol 3'-kinase (PI3K) and phpspholipase C (PLC), by blocking those pathways using inhibitors of each kinase, and also investigated the effects on the phosphorylation of myosin light chain (MLC). RESULTS: Gefitinib inhibited proliferation in most of these cell lines. MDA-MB231 was shown to be resistant. Furthermore, proliferation of MDA-MB231 cells was not affected by EGF stimulation, but migration of MDA-MB231 cells was significantly inhibited. PI3K and PLC inhibitors blocked EGF-stimulated cell migration and MLC phosphorylation, but the MEK inhibitor did not influence cell migration. CONCLUSIONS: Gefitinib has an anti-migratory effect on MDA-MB231 that results in an anti-proliferative effect. PI3K and PLC are important for the migration of MDA-MB231 cells, and gefitinib may inhibit migration by blocking these signalling pathways.     

Antitumor effect of gefitinib ('Iressa') on esophageal squamous cell carcinoma cell lines in vitro and in vivo

High expression of epidermal growth factor receptor (EGFR) is thought to be correlated with cell proliferation, invasion, metastasis, resistance to chemoradiotherapy, and poor prognosis in various kinds of human cancers. Blockade of EGFR signal transduction can be a promising strategy for cancer therapy. Approximately 40-70% of esophageal squamous cell carcinomas (ESCCs) show high expression of EGFR. In this study, we examined the antitumor effect of gefitinib, an EGFR tyrosine kinase inhibitor, against ESCC cells in vitro and in vivo. In three ESCC cell lines (TE8, T.T and T.Tn), cell proliferation had been inhibited in a dose-dependent manner and IC(50) values (respectively, 8.49, 18.9 and 17.3muM). Gefitinib inhibited EGF-induced autophosphorylation of EGFR and its downstream signaling pathways, Ras/Raf/MAPK and PI3K/Akt, and caused G(1) arrest of cell cycle and apoptosis confirmed with flow cytometry. We examined the effect of gefitinib on nude mice bearing established TE8 and T.T xenografts. Gefitinib (100 or 200mg/kg once-daily, p.o.) showed antitumor activity in a dose-dependent manner, resulting in a significantly improved survival of treated mice as compared with untreated mice. Immunohistochemical examination of the harvested tumor was performed to examine the status of phosphorylated EGFR, PCNA, Factor VIII and apoptosis. We found inhibition of EGFR phosphorylation, cell cycle arrest (by PCNA staining), decrease of microvessel density (Factor VIII) and induction of apoptosis by TUNEL staining. In conclusion, our findings demonstrate that gefitinib is effective for growth inhibition of ESCC cell lines in vitro and in vivo and suggest that gefitinib may be one of the new therapeutic options for ESSC.     

Role of GRB2‐associated binder 1 in epidermal growth factor receptor‐induced signaling in head and neck squamous cell carcinoma

The epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of head and neck squamous cell carcinoma (HNSCC). Despite the high expression of EGFR in HNSCC, EGFR inhibitors have only limited success as monotherapy. The Grb2‐associated binder (GAB) family of adaptor proteins acts as docking/scaffolding molecules downstream of tyrosine kinase receptors. We hypothesized that GAB1 may amplify EGFR‐induced signaling in HNSCCs and therefore could play a role in the reduced sensitivity of HNSCC to EGFR inhibitors. We used representative human HNSCC cell lines overexpressing wild type EGFR, and expressing GAB1 but not GAB2. We demonstrated that baseline Akt and MAPK signaling were reduced in HNSCC cells in which GAB1 expression was reduced. Furthermore, the maximal EGF‐induced activation of the Akt and MAPK pathway was reduced and delayed, and the duration of the EGF‐induced activation of these pathways was reduced in cells with GAB1 knock‐down. In agreement with this, HNSCC cells in which GAB1 levels were reduced showed an increased sensitivity to the EGFR inhibitor gefitinib. Our work demonstrates that GAB1 plays an important role as part of the mechanism of by which EGFR induces induced activation of the MAPK and AKT pathway. Our results identify GAB1 as an amplifier of the EGFR‐initiated signaling, which may also interfere with EGFR degradation. These findings support the emerging notion that reducing GAB1 function may sensitize HNSCC to EGFR inhibitors, hence representing a new therapeutic target HNSCC treatment in combination with EGFR targeting agents.     

Gefitinib (ZD1839) increases the efficacy of cisplatin in ovarian cancer cells

We examined the effect of gefitinib (ZD1839), a selective epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, on cytotoxicity to cisplatin, EGFR downstream signaling, apoptosis and the association between the inhibition of DNA repair by gefitinib and the expression of DNA-dependent protein kinase (DNA-PK) using three ovarian cancer cell lines. In the presence of gefitinib, cisplatin-induced growth inhibition and apoptosis were significantly enhanced in Caov-3 and RMG-1 cells, which express EGFR, and in A2780, which lacks EGFR but expresses HER-2. Gefitinib significantly inhibited the cisplatin-induced ERK and Akt activation in Caov-3 and RMG-1 cells but not in A2780 cells. In all three cell lines, there was delayed repair of DNA intrastrand cross-links damaged by cisplatin used in combination with gefitinib, compared with cisplatin alone. The reduction in DNA-PK levels persisted when cells were exposed to combinations of cisplatin and gefitinib in all cell lines. Moreover, the delayed repair was cancelled by anti-HER2 small-interfering RNA transfection in A2780 cells. These results suggest that combination therapy with cisplatin and gefitinib may increase the therapeutic efficacy of cisplatin by blocking EGFR downstream signaling and/or inhibiting DNA repair in ovarian cancer.     

SU11274逆转肝细胞生长因子诱导不同EGFR基因型非小细胞肺癌细胞株对吉非替尼耐药

背景与目的：肝细胞生长因子(hepatocyte growth factor,HGF)诱导敏感非小细胞肺癌(nonsmall cell lung cancer,NSCLC)细胞对表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinase inhibitor,EGFR-TKI)耐药,其机制与c-Met激活有关。本研究探讨c-Met抑制剂SU11274逆转HGF诱导的不同EGFR基因型NSCLC细胞株对吉非替尼耐药及逆转耐药机制。方法：选择人NSCLC细胞株PC9(EGFR突变型)、H292(EGFR野生型)和A549(EGFR野生型),应用吉非替尼和SU11274单独或联合作用于HGF诱导的细胞株。实验分为6组：C组(不加药对照组)、H组(HGF处理组)、G组(吉非替尼处理组)、S(SU11274处理组)、HG组(HGF+吉非替尼处理组)和HGS组(HGF+吉非替尼+SU11274处理组)。MTT法检测对细胞增殖的影响,流式细胞术检测细胞凋亡的影响;应用蛋白质印迹法(Western blot)检测细胞中c-Met及其下游通道Stat3、Akt和Erk1/2蛋白表达水平。结果：吉非替尼对3种细胞的生长抑制作用均呈浓度依赖性,HGF处理能够缓解吉非替尼的增殖抑制作用(P<0.05);不同浓度吉非替尼联合SU11274作用于HGF诱导细胞时,3种细胞株存活率比吉非替尼单独作用于HGF诱导细胞时明显降低(P<0.05);HGS组的细胞凋亡比HG组明显增加(P<0.05);HGS组的c-Met、Stat3、Akt和Erk1/2活化蛋白量比HG组明显减少。结论：c-Met抑制剂SU11274可逆转HGF诱导的不同EGFR基因型NSCLC细胞株对吉非替尼耐药,其机制可能与抑制HGF活化的c-Met及其下游通道蛋白表达有关。     

Molecular basis for sensitivity and acquired resistance to gefitinib in HER2-overexpressing human gastric cancer cell lines derived from liver metastasis

Gastric cancer metastasised to the liver was found to overexpress HER2 at a significantly higher incidence than primary gastric cancers. The purpose of the present study was to investigate the possibility of molecular therapy targeting HER2 overexpression in gastric cancer liver metastasis. We developed three new HER2-overexpressing gastric cancer cell lines (GLM-1, GLM-2, GLM-4) without epidermal growth factor receptor (EGFR) mutations derived from such liver metastasis, two of which had HER2 gene amplifications. All these GLM series of cell lines were highly sensitive to gefitinib in vitro, a specific inhibitor of EGFR tyrosine kinase (Iressa) rather than anti-HER2 antibody trastuzumab (Herceptin), whereas most of the HER2 low-expressing counterparts were not. In these HER2-overexpressing GLM series, protein kinase B (Akt), but not extracellular signal-regulated kinase 1/2 (ERK1/2), was constitutively phosphorylated, and gefitinib efficiently inhibited this Akt phosphorylation, induced strong apoptosis in vitro and exhibited antitumour activity in tumour xenografts in nude mice. This gefitinib-mediated antitumour effect in xenograft was significantly potentiated by trastuzumab treatment. On the other hand, gefitinib-resistant cells (GLM-1R) exhibited increased EGFR expression, followed by constitutive activation of mitogen-activated protein kinase (MAPK) pathway. These results suggest that the antitumour effect of gefitinib is due to the effective inhibition of HER2-driven constitutive activation of phosphatidylinositol-3-kinase (PI3K)/Akt pathway, and that the acquired resistance to gefitinib is due to the constitutive activation of Ras/MAPK pathway in compensation for PI3K/Akt pathway. Gastric cancer liver metastasis with HER2 overexpression would be a potential molecular target for gefitinib and trastuzumab.     

Mutations of the epidermal growth factor receptor gene in gastrointestinal tract tumor cell lines

The epidermal growth factor receptor (EGFR) is commonly overexpressed in many human tumors including gastrointestinal tract tumors. Gefitinib is a selective inhibitor of EGFR tyrosine kinase, and blocks several signal transduction pathways including those involved in tumor cell proliferation, angiogenesis and metastasis. Recent mutational and biological studies have suggested that mutations in the tyrosine kinase domain of the EGFR gene are well correlated with the response to gefitinib, and that these mutations are frequently observed in non-small cell lung cancers affecting women, East Asians and non-smokers. This led us to speculate that EGFR gene mutations may occur frequently in gastrointestinal tract carcinomas (GITCs) because overexpression is observed in these tumor types. To investigate EGFR mutations in GICTs, we studied 11 esophageal, 6 gastric, and 12 colorectal cancer cell lines. We found a missense mutation in a gastric cancer cell line, and 10 single nucleotide polymorphisms. The occurrence of rare mutations in the tyrosine kinase domain of the EGFR gene suggests that gefitinib is unlikely to be reliable as single-drug therapy for GITCs.     

Epidermal growth factor receptor (EGFR) is overexpressed in anaplastic thyroid cancer, and the EGFR inhibitor gefitinib inhibits the growth of anaplastic thyroid cancer.

PURPOSE: No effective treatment options currently are available to patients with anaplastic thyroid cancer (ATC), resulting in high mortality rates. Epidermal growth factor (EGF) has been shown to play a role in the pathogenesis of many types of cancer, and its receptor (EGFR) provides an attractive target for molecular therapy. EXPERIMENTAL DESIGN: The expression of EGFR was determined in ATC in vitro and in vivo and in human tissue arrays of ATC. We assessed the potential of the EGFR inhibitor gefitinib ("Iressa," ZD1839) to inhibit EGFR activation in vitro and in vivo, inhibit ATC cellular proliferation, induce apoptosis, and reduce the growth of ATC cells in vivo when administered alone and in combination with paclitaxel. RESULTS: EGFR was overexpressed in ATC cell lines in vitro and in vivo and in human ATC specimens. Activation of EGFR by EGF was blocked by the addition of gefitinib. In vitro studies showed that gefitinib greatly inhibited cellular proliferation and induced apoptosis in ATC cell lines and slowed tumor growth in a nude mouse model of thyroid carcinoma cells injected subcutaneously. CONCLUSIONS: ATC cells consistently overexpress EGFR, rendering this receptor a potential target for molecular therapy. Gefitinib effectively blocks activation of EGFR by EGF, inhibits ATC cellular proliferation, and induces apoptosis in vitro. Our in vivo results show that gefitinib has significant antitumor activity against ATC in a subcutaneous nude mouse tumor model and therefore is a potential candidate for human clinical trials.