N-Glycan fucosylation of epidermal growth factor receptor modulates receptor activity and sensitivity to epidermal growth factor receptor tyrosine kinase inhibitor

The glycosylation of cell surface proteins is important for cancer biology processes such as cellular proliferation or metastasis. α1,6-Fucosyltransferase (FUT8) transfers a fucose residue to n -linked oligosaccharides on glycoproteins. Herein, we study the effect of fucosylation on epidermal growth factor receptor (EGFR) activity and sensitivity to an EGFR-specific tyrosine kinase inhibitor (EGFR-TKI). The increased fucosylation of EGFR significantly promoted EGF-mediated cellular growth, and the decreased fucosylation by stable FUT8 knockdown weakened the growth response in HEK293 cells. The overexpression of FUT8 cells were more sensitive than the control cells to the EGFR-TKI gefitinib, and FUT8 knockdown decreased the sensitivity to gefitinib. Finally, to examine the effects in a human cancer cell line, we constructed stable FUT8 knockdown A549 cells, and found that these cells also decreased EGF-mediated cellular growth and were less sensitive than the control cells to gefitinib. In conclusion, we demonstrated that the modification of EGFR fucosylation affected EGF-mediated cellular growth and sensitivity to gefitinib. Our results provide a novel insight into how the glycosylation status of a receptor may affect the sensitivity of the cell to molecular target agents. ( Cancer Sci 2008; 99: 1611–1617)     

Oncogenic fingerprint of epidermal growth factor receptor pathway and emerging epidermal growth factor receptor blockade resistance in colorectal cancer

Epidermal growth factor receptor (EGFR) has been an attractive target for treatment of epithelial cancers, including colorectal cancer (CRC). Evidence from clinical trials indicates that cetuximab and panitumumab (anti-EGFR monoclonal antibodies) have clinical activity in patients with metastatic CRC. The discovery of intrinsic EGFR blockade resistance in Kirsten RAS (KRAS)-mutant patients led to the restriction of anti-EGFR antibodies to KRAS wild-type patients by Food and Drug Administration and European Medicine Agency. Studies have since focused on the evaluation of biomarkers to identify appropriate patient populations that may benefit from EGFR blockade. Accumulating evidence suggests that patients with mutations in EGFR downstream signaling pathways including KRAS, BRAF, PIK3CA and PTEN could be intrinsically resistant to EGFR blockade. Recent whole genome studies also suggest that dynamic alterations in signaling pathways downstream of EGFR leads to distinct oncogenic signatures and subclones which might have some impact on emerging resistance in KRAS wild-type patients. While anti-EGFR monoclonal antibodies have a clear potential in the management of a subset of patients with metastatic CRC, further studies are warranted to uncover exact mechanisms related to acquired resistance to EGFR blockade.     

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.     

Cytokine regulation by epidermal growth factor receptor inhibitors and epidermal growth factor receptor inhibitor associated skin toxicity in cancer patients

AimEpidermal growth factor receptor inhibitor (EGFRI) induced skin toxicity has a prognostic value suggesting skin toxicity can be a useful surrogate marker for successful epidermal growth factor receptor (EGFR) inhibition, improved response and survival. But the pathophysiology of EGFRI induced skin toxicity remains elusive. However the involvement of immunological mechanisms has been speculated. This study investigates the possible underlying mechanism of EGFR inhibition associated cytokine production in keratinocytes as well as in patients after treatment with epidermal growth factor receptor inhibitors (EGFRIs).MethodsNormal human epidermal keratinocytes (NHEK) were incubated for 2 and 24 h with different concentrations of EGFRI (erlotinib) for Western blot analysis and cytokine expression analysis, respectively. Inhibition of EGFR, extracellular-signal-regulated kinase 1/2 (Erk 1/2) and c-Jun was examined by Western blot analysis. Cytokine concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in the NHEK cell supernatant and also in the serum of 186 cancer patients who are followed up for EGFRI induced skin rash.ResultsA significant inhibitory effect of EGFRI was seen on EGFR (Y845), Erk 1/2 and c-Jun in a dose dependent manner. Further downstream, increased CC-chemokine ligand 2 (CCL2), CC-chemokine ligand 5 (CCL5) and decreased interleukin-8 (IL-8) or CXCL8 expression was observed in keratinocytes. In EGFRI treated patients, low levels of serum CXCL8 corresponding to stronger EGFR inhibition were associated with a higher grade of skin toxicity (p = 0.0016) and a prolonged overall survival (p = 0.018).ConclusionsThe results obtained in this study indicate that EGFRI can regulate cytokines by modulating EGFR signalling pathway in keratinocytes. Moreover, serum levels of CXCL8 in EGFRI treated patients may be important for individual EGFRI induced skin toxicity and patient’s survival.     

Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer

Recent discovery of mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene in lung adenocarcinoma greatly stimulated biomarker research on predictive factors for EGFR tyrosine kinase inhibitors (TKI), such as gefitinib and erlotinib. Although patients with activating mutations of the EGFR generally respond to EGFR TKIs very well, it is natural to assume that there is no sole determinant, considering great complexity and redundancy of the EGFR pathway. Subsequently, roles of different types of EGFR mutations or mutations of genes that are members of the EGFR pathway such as KRAS and HER2 have been evaluated. In this review, we summarize the recent findings about how mutations of the EGFR and related genes affect sensitivity to EFGR-TKIs. We also discuss molecular mechanisms of acquired resistance to EGFR-TKIs that is almost inevitable in EGFR-TKI therapy. The door for genotype-based treatment of lung cancer is beginning to open, and through these efforts, it will be possible to slow the progression of lung cancer and eventually, to decrease mortality from lung cancer. ( Cancer Sci 2007; 98: 1817–1824)     

Amplification and overexpression of the epidermal growth factor receptor gene in human renal-cell carcinoma

We examined 22 cases of renal-cell carcinoma (RCC) for structural alterations of the epidermal growth factor receptor (EGFR) gene and found gene amplification in one case of high-stage-high-grade RCC. Dot blot analysis of the total RNA from tumorous and normal kidney tissues revealed overexpression of the EGFR gene in 12 of 20 (60%) cases of RCC. The highest expression was observed in the gene-amplified case. No correlation was observed between the level of EGFR mRNA and tumor stage or grade. Northern blot analysis revealed normal 10- and 5.6-kb EGFR mRNA bands in RCC. Our data indicate that gene amplification of the EGFR gene is one of the molecular mechanisms of its overexpression in a subset of RCCs.     

Kinetic analysis of epidermal growth factor receptor somatic mutant proteins shows increased sensitivity to the epidermal growth factor receptor tyrosine kinase inhibitor, erlotinib

We show that two commonly occurring epidermal growth factor receptor (EGFR) somatic mutations, L858R and an in-frame deletion mutant, Del(746-750), exhibit distinct enzymatic properties relative to wild-type EGFR and are differentially sensitive to erlotinib. Kinetic analysis of the purified intracellular domains of EGFR L858R and EGFR Del(746-750) reveals that both mutants are active but exhibit a higher K(M) for ATP and a lower K(i) for erlotinib relative to wild-type receptor. When expressed in NR6 cells, a cell line that does not express EGFR or other ErbB receptors, both mutations are ligand dependent for receptor activation, can activate downstream EGFR signaling pathways, and promote cell cycle progression. As expected from the kinetic analysis, the EGFR Del(746-752) is more sensitive to erlotinib inhibition than the EGFR L858R mutant. Further characterization shows that these mutations promote ligand-dependent and anchorage-independent growth, and cells harboring these mutant receptors form tumors in immunocompromised mice. Analysis of tumor lysates reveals that the tumorigenicity of the mutant EGFR cell lines may be due to a differential pattern of mutant EGFR autophosphorylation as compared with wild-type receptor. Significant inhibition of tumor growth, in mice harboring wild-type EGFR receptors, is only observed at doses of erlotinib approaching the maximum tolerated dose for the mouse. In contrast, the growth of mutant tumors is inhibited by erlotinib treatment at approximately one third the maximum tolerated dose. These findings suggest that EGFR somatic mutations directly influence both erlotinib sensitivity and cellular transformation.     

ERK phosphorylation predicts synergism between gemcitabine and the epidermal growth factor receptor inhibitor AG1478

Background

Clinical trials combining epidermal growth factor receptor (EGFR) inhibitors with gemcitabine-based chemotherapy in non-small cell lung cancer (NSCLC) have not produced a survival advantage. This may be caused by antagonism between the two drugs or mutations that promote such, possibly RAS mutation. Furthermore, ERK, a critical growth regulator downstream of RAS, may play a role. This study aimed to explore the relationship between ERK, synergy/antagonism and cell cycle arrest in combination treatment.

Methods

A549 (mutant KRAS), H322 (wildtype KRAS) and siRNA-mediated KRAS knockdown A549 were treated with gemcitabine and/or the EGFR inhibitor AG1478 and analyzed with median effect analysis. Cell cycle distribution and ERK phosphorylation were assessed using flow cytometry and ELISA, respectively. Effect on cytotoxicity after ERK inhibition by U0126 was also assessed.

Results

Cytotoxic interaction was dose dependent with antagonism at high dose AG1478. G1 arrest was observed with both high dose AG1478 and high dose gemcitabine and therefore was inconsistently associated with antagonism. Furthermore, ERK phosphorylation was increased by gemcitabine and its suppression by AG1478 was related to antagonism particularly in H322. ERK's effect in antagonism was further confirmed by using U0126. Greater antagonism was observed in the KRAS mutant cell line and KRAS knockdown by siRNA resulted in increased sensitivity to AG1478 as well as combination treatment.

Conclusion

Our findings are consistent with a model in which ERK phosphorylation favors synergy and the outcome depends on the balance between gemcitabine-induced and AG1478-inhibited ERK phosphorylation. KRAS mutation confers resistance to AG1478 as well as combination treatment.     

Targeting epidermal growth factor receptor/human epidermal growth factor receptor 2 signalling pathway by a dual receptor tyrosine kinase inhibitor afatinib for radiosensitisation in murine bladder carcinoma

Given the promising control of bladder cancer achieved by combined chemotherapy/radiotherapy with selective transurethral resection, obstacles remain to the treatment of unresectable bladder cancer. The aim of this study was to determine whether targeting epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) can radiosensitise a murine bladder tumour (MBT-2) cell line. Cell survival, expression of signal proteins and cell cycle changes in MBT-2 cells treated in vitro and in vivo with afatinib, an irreversible EGFR/HER2 inhibitor, plus radiotherapy were investigated by colony formation assay, Western blot assay and flow cytometry, respectively. Ectopic xenografts were established by subcutaneous injection of MBT-2 cells in C3H/HeN mice. Mice were randomised into 4 groups to receive afatinib (10 mg/kg/day on day 1–7) and/or radiotherapy (15 Gy on day 4). Positron emission tomography (PET) on day 8 was used to evaluate the early treatment response. Afatinib (200–1000 nM) increased cell killing by radiation (0–10 Gy). Pre-treatment of irradiated cells with afatinib inhibited radiation-activated HER2 and EGFR phosphorylation. As compared to either treatment alone, the combination increased the level of the cleavage form of poly (ADP-ribose) polymerase, the expression of phospho-γH2AX and the percentage of cells in subG1 phase (indicating enhanced induction of apoptosis), and decreased tumour metabolism and inhibited tumour growth by 64%. Afatinib has therapeutic value as a radiosensitiser of murine bladder cancer cells. The synergism between afatinib and radiation likely enhances DNA damage, leading to increased cell apoptosis.     

Involvement of epidermal growth factor receptor overexpression in the promotion of breast cancer brain metastasis

BACKGROUND:

Brain‐metastatic breast cancer (BMBC) is increasing and poses a severe clinical problem because of the lack of effective treatments and because the underlying molecular mechanisms are largely unknown. Recent work has demonstrated that deregulation of epidermal growth factor receptor (EGFR) may correlate with BMBC progression. However, the exact contribution that EGFR makes to BMBC remains unclear.

METHODS:

The role of EGFR in BMBC was explored by serial analyses in a brain‐trophic clone of human MDA‐MB‐231 breast carcinoma cells (231‐BR cells). EGFR expression was inhibited by stable short‐hairpin RNA transfection or by the kinase inhibitor erlotinib, and it was activated by heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF). Cell growth and invasion activities also were analyzed in vitro and in vivo.

RESULTS:

EGFR inhibition or activation strongly affected 231‐BR cell migration/invasion activities as assessed by an adhesion assay, a wound‐healing assay, a Boyden chamber invasion assay, and cytoskeleton staining. Also, EGFR inhibition significantly decreased brain metastases of 231‐BR cells in vivo. Surprisingly, changes to EGFR expression affected cell proliferation activities less significantly as determined by a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, an anchorage‐independent growth assay, and cell cycle analysis. Immunoblot analysis suggested that EGFR drives cells' invasiveness capability mainly through phosphoinositide 3‐kinase/protein kinase B and phospholipase C γ downstream pathways. In addition, EGFR was involved less in proliferation because of the insensitivity of the downstream mitogen‐activated protein kinase pathway.

CONCLUSIONS:

The current results indicated that EGFR plays more important roles in cell migration and invasion to the brain than in cell proliferation progression on 231‐BR cells, providing new evidence of the potential value of EGFR inhibition in treating BMBC. Cancer 2012. © 2012 American Cancer Society.     

Targeting epidermal growth factor receptor in lung cancer

Among the most promising agents in clinical development to treat non-small-cell lung cancer (NSCLC) are the epidermal growth factor receptor (EGFR) targeting agents. A series of recent studies have demonstrated the activity of anti-EGFR targeted therapies for NSCLC. In advanced NSCLC that is refractory to chemotherapy, antitumor responses have been reported with EGFR tyrosine kinase inhibitors (ZD1839 and OSI-774). The role of ZD1839 and OSI-774 as possible additions to standard chemotherapy in the first-line setting has also been evaluated, and the studies conducted to date should respond to the question of whether these compounds could provide a survival benefit. Other areas of research involve looking at the role of EGFR tyrosine kinase inhibitors in the neoadjuvant treatment of stage III NSCLC and the planning of chemoprevention studies. These exciting results and plans are further complemented by an emerging number of compounds in clinical development, including both monoclonal antibodies (ie, IMC-C225) and other tyrosine kinase inhibitors, directed at the EGFR.     

Epidermal growth factor receptor lacking C-terminal autophosphorylation sites retains signal transduction and high sensitivity to epidermal growth factor receptor tyrosine kinase inhibitor

Constitutively active mutations of epidermal growth factor receptor (EGFR) (delE746_A750) activate downstream signals, such as ERK and Akt, through the phosphorylation of tyrosine residues in the C-terminal region of EGFR. These pathways are thought to be important for cellular sensitivity to EGFR tyrosine kinase inhibitors (TKI). To examine the correlation between phosphorylation of the tyrosine residues in the C-terminal region of EGFR and cellular sensitivity to EGFR TKI, we used wild-type (wt) EGFR, as well as the following constructs: delE746_A750 EGFR; delE746_A750 EGFR with substitution of seven tyrosine residues to phenylalanine in the C-terminal region; and delE746_A750 EGFR with a C-terminal truncation at amino acid 980. These constructs were transfected stably into HEK293 cells and designated HEK293/Wt, HEK293/D, HEK293/D7F, and HEK293/D-Tr, respectively. The HEK293/D cells were found to be 100-fold more sensitive to EGFR TKI (AG1478) than HEK293/Wt. Surprisingly, the HEK293/D7F and HEK293/D-Tr cells, transfected with EGFR lacking the C-terminal autophosphorylation sites, retained high sensitivity to EGFR TKI. In these three high-sensitivity cells, the ERK pathway was activated without ligand stimulation, which was inhibited by EGFR TKI. In addition, although EGFR in the HEK293/D7F and HEK293/D-Tr cells lacked significant tyrosine residues for EGFR signal transduction, phosphorylation of Src homology and collagen homology (Shc) was spontaneously activated in these cells. Our results indicate that tyrosine residues in the C-terminal region of EGFR are not required for cellular sensitivity to EGFR TKI, and that an as-yet-unknown signaling pathway of EGFR may exist that is independent of the C-terminal region of EGFR. ( Cancer Sci 2009; 100: 552–557)     

Relationship between activation of epidermal growth factor receptor and cell dissociation in pancreatic cancer

In our previous investigations, mitogen-activated protein kinase kinase 2 (MEK2)/extracellular signal-regulated kinase 2 (ERK2) signaling pathway was found to be correlated with the cell dissociation induced by dissociation factor (DF) in pancreatic cancer cells. In this study, the expressions of epidermal growth factor receptor (EGFR), phosphorylated EGFR (p-EGFR), and its downstream kinases MEK1/2 and ERK1/2, were analyzed to clarify the regulatory mechanism of cell dissociation in pancreatic cancer cells. Two hamster (PC-1.0 and PC-1) and two human (AsPC-1 and Capan-2) pancreatic cancer cell lines were used. Immunocytochemical study was performed using anti-EGFR, p-EGFR, phosphorylated MEK1/2 (p-MEK1/2), and phosphorylated ERK1/2 (p-ERK1/2) antibodies. DF-treatment markedly induced the expressions of EGFR, p-EGFR, p-MEK1/2, p-ERK1/2, as well as the dissociation of cell colonies in PC-1 and Capan-2 cells. In contrast, AG1478 (an EGFR inhibitor) treatment significantly induced the cell aggregation in PC-1.0 and AsPC-1 cells which usually grew as single cells, but strongly suppressed the expressions of EGFR, p-EGFR, p-MEK1/2, and p-ERK1/2. These observations demonstrate that activation of EGFR is closely involved in cell dissociation in pancreatic cancer through activating MEK/ERK signaling pathway.     

Blockade of vascular endothelial growth factor receptor and epidermal growth factor receptor signaling for therapy of metastatic human pancreatic cancer

We determined whether concurrent blockage of vascular endothelial growth factor (VEGF) receptor and epidermal growth factor (EGF) receptor signaling by two novel tyrosine kinase inhibitors, PTK 787 and PKI 166, respectively, can inhibit angiogenesis and, hence, the growth and metastasis of human pancreatic carcinoma in nude mice. Highly metastatic human pancreatic carcinoma L3.6pl cells were injected into the pancreas of nude mice. Seven days later, groups of mice began receiving oral doses of PTK 787 and PKI 166 three times weekly. Some groups of mice also received i.p. injections of gemcitabine twice a week. The mice were necropsied when the control mice became moribund. Treatment with PTK 787 and PKI 166, with gemcitabine alone, or with the combination of PTK 787, PKI 166, and gemcitabine produced 69, 50, and 97% reduction in the volume of pancreatic tumors, respectively. Administration of protein tyrosine kinase inhibitors and gemcitabine also significantly decreased the incidence of lymph node and liver metastasis. The therapeutic efficacy directly correlated with a decrease in circulating proangiogenic molecules (VEGF, interleukin-8), a decrease in microvessel density, a decrease in proliferating cell nuclear antigen staining, and an increase in apoptosis of tumor cells and endothelial cells. Therapies produced by combining gemcitabine with either PKI 166 or PTK 787 were similar to those produced by combining gemcitabine with both PKI 166 and PTK 787. These results suggest that blockade of either epidermal growth factor receptor or VEGF receptor signaling combined with chemotherapy provides an effective approach to the therapy of pancreatic cancer.     

Enhancing oxaliplatin-based regimens in colorectal cancer by inhibiting the epidermal growth factor receptor pathway

In the past several years, significant advances in the treatment of colorectal cancer (CRC) have been made. With the introduction of irinotecan and oxaliplatin combined with infusional 5-fluororuracil, survival times for patients with metastatic CRC have nearly doubled. With the incorporation of biologic agents that target the vascular endothelial growth factor and epidermal growth factor (EGF) pathways, additional improvements in response, progression-free survival, and overall survival have been seen in patients with advanced, metastatic disease. This article reviews the impact of EGF receptor inhibitors on improving survival in CRC when combined with oxaliplatin-containing regimens.     

Cortactin overexpression inhibits ligand-induced down-regulation of the epidermal growth factor receptor

Ligand-induced receptor down-regulation by endocytosis is a critical process regulating the intensity and duration of receptor tyrosine kinase signaling. Ubiquitylation of specific receptor tyrosine kinases, for example, the epidermal growth factor receptor (EGFR) by the E3 ubiquitin ligase c-Cbl, provides a sorting signal for lysosomal degradation and leads to termination of receptor signaling. Cortactin, which couples the endocytic machinery to dynamic actin networks, is encoded by EMS1, a gene commonly amplified in breast and head and neck cancers. One mechanism whereby cortactin overexpression contributes to tumor progression is by enhancing tumor cell invasion and metastasis. However, in this study, we show that overexpression of cortactin in HeLa cells markedly inhibits ligand-induced down-regulation of the EGFR. This is independent of alterations in receptor autophosphorylation and correlates with impaired c-Cbl phosphorylation and association with the EGFR, reduced EGFR ubiquitylation, and sustained EGF-induced extracellular signal-regulated kinase activation. Furthermore, analysis of a panel of head and neck squamous cell carcinoma (HNSCC) cell lines revealed that cortactin overexpression is associated with attenuated ligand-induced EGFR down-regulation. Importantly, RNAi-mediated reduction of cortactin expression in an 11q13-amplified HNSCC cell line accelerates EGFR degradation. This represents the first demonstration of modulation of growth factor receptor signaling by cortactin. Moreover, enhanced EGFR signaling due to cortactin overexpression may provide an alternative explanation for EMS1 gene amplification in human cancers.     

Expression of epidermal growth factor receptor gene in cultured human lung cancer cells

The expression and organization of epidermal growth factor (EGF) receptor gene in cultured human lung cancer cell lines (5 adenocarcinomas, 3 squamous cell carcinomas, 2 small cell carcinomas and 1 large cell carcinoma) have been studied. Two (PC-8 and PC-9) of the adenocarcinomas overproduced EGF receptor mRNA and protein, and exhibited gene amplification, the magnitude of which was comparable to that of A431 cells. Six cell lines (3 adenocarcinomas, 2 squamous cell carcinomas and 1 small cell carcinoma) expressed EGF receptor gene and its product to a significant level without gene amplification, and the other three cell lines were found to be negative as regards expression.