The Effect of Arylquinoline-Based EGFR and FAK Kinase Inhibitors on the Activity of Major Cytochrome P450 Enzymes.

Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis. Anti-phosphotyrosine and anti-EGFr affinity chromatography, isotope-coded muLC-MS/MS, and immunoblot methods were combined to describe and measure signaling networks associated with EGF receptor activation and pharmacological inhibition. The squamous carcinoma cell line HN5, which overexpresses EGF receptor and displays sustained receptor kinase activation, was used as a model system, where pharmacological inhibition of EGF receptor kinase by erlotinib markedly reduced auto and substrate phosphorylation, Src family phosphorylation at EGFR Y845, while increasing total EGF receptor protein. Diverse sets of known and poorly described functional protein classes were unequivocally identified by affinity selection, comprising either proteins tyrosine phosphorylated or complexed therewith, predominantly through EGF receptor and Src family kinases, principally 1) immediate EGF receptor signaling complexes (18%); 2) complexes involved in adhesion and cell-cell contacts (34%); and 3) receptor internalization and degradation signals. Novel and known phosphorylation sites could be located despite the complexity of the peptide mixtures. In addition to interactions with multiple signaling adaptors Grb2, SHC, SCK, and NSP2, EGF receptors in HN5 cells were shown to form direct or indirect physical interactions with additional kinases including ACK1, focal adhesion kinase (FAK), Pyk2, Yes, EphA2, and EphB4. Pharmacological inhibition of EGF receptor kinase activity by erlotinib resulted in reduced phosphorylation of downstream signaling, for example through Cbl/Cbl-B, phospholipase Cgamma (PLCgamma), Erk1/2, PI-3 kinase, and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, and plakophillin were down-regulated by transient EGF stimulation suggesting a complex balance between growth factor induced kinase and phosphatase activities in the control of cell adhesion complexes. The functional interactions between IGF-1 receptor, lysophosphatidic acid (LPA) signaling, and EGF receptor were observed, both direct and/or indirectly on phospho-Akt, phospho-Erk1/2, and phospho-ribosomal S6.

Here we describe a liquid chromatography/mass spectrometry (LC/MS) method with automated online extraction (LC/LC/MS) to simultaneously determine the in vitro inhibitory potency of herbal extracts on six major human drug-metabolising cytochrome P450 enzymes. Substrates were incubated with a commercially available mixture of CYP1A2/2C8/2C9/2C19/2D6 and 3A4 from baculovirus-infected insect cells and the resulting metabolites were quantified with LC/LC/MS using electrospray ionisation in the selected ion monitoring mode. Consistent inhibitory activities were obtained for known inhibitors and plant extracts using the enzyme/substrate cocktail and the individual enzymes/substrates. Popular herbal remedies including devil's claw root (Harpagophytum procumbens), feverfew herb (Tanacetum parthenium), fo-ti root (Polygonum multiflorum), kava-kava root (Piper methysticum), peppermint oil (Mentha piperita), eucalyptus oil (Eucalyptus globulus), red clover blossom (Trifolium pratense) and grapefruit juice (GJ; Citrus paradisi) could be identified as inhibitors of the applied CYP enzymes with IC(50) values between 20 and 1000 microg/mL.

In this study, we present multiple lines of evidence to support a critical role for heparin-bound EGF (epidermal growth factor)-like growth factor (HB-EGF) and tumor necrosis factor-alpha-converting enzyme (TACE) (ADAM17) in the transactivation of EGF receptor (EGFR), ERK phosphorylation, and cellular proliferation induced by the 5-HT(2A) receptor in renal mesangial cells. 5-hydroxy-tryptamine (5-HT) resulted in rapid activation of TACE, HB-EGF shedding, EGFR activation, ERK phosphorylation, and longer term increases in DNA content in mesangial cells. ERK phosphorylation was attenuated by 1) neutralizing EGFR antibodies and the EGFR kinase inhibitor, AG1478, 2) neutralizing HB-EGF, but not amphiregulin, antibodies, heparin, or CM197, and 3) pharmacological inhibitors of matrix-degrading metalloproteinases or TACE small interfering RNA. Exogenously administered HB-EGF stimulated ERK phosphorylation. Additionally, TACE was co-immunoprecipitated with HB-EGF. Small interfering RNA against TACE also blocked 5-HT-induced increases in ERK phosphorylation, HB-EGF shedding, and DNA content. In aggregate, this work supports a pathway map that can be depicted as follows: 5-HT --> 5-HT(2A) receptor --> TACE --> HB-EGF shedding --> EGFR --> ERK --> increased DNA content. To our knowledge, this is the first time that TACE has been implicated in 5-HT-induced EGFR transactivation or in proliferation induced by a G protein-coupled receptor in native cells in culture.

Both 9‐a (sulphonamide derivative) and WES‐1 (coumarin derivative) were designed and synthesized as potential selective Carbonic Anhydrase Inhibitors, and are currently being tested for anti‐cancer activity. This study was undertaken to investigate their potential inhibitory effects on the major Cytochrome P450 (CYP) drug metabolizing enzymes in human liver microsomes using specific CYP probe substrates and liquid chromatography‐tandem mass spectrometry. 9a potently inhibited CYP2E1‐catalyzed chlorzoxazone‐6′‐hydrolylation and CYP2C9‐catalyzed tolbutamide‐4′‐hydrolylation with IC50 values of 0.05 and 0.7 μM, respectively. CYP2A6‐catalyzed coumarin‐7′‐hydroxylation and CYP3A4‐catalyzed testosterone‐6β‐hydroxylation were moderately inhibited by 9‐a with IC50 values of 2.7 and 12 μM, respectively. The rest of the tested CYP enzymes were weakly or negligibly inhibited by 9‐a. WES‐1, on the other hand, demonstrated weak inhibitory effects on CYP2C19‐mediated [S]‐mephenytoin‐4′‐hydrolylation, CYP2C9 activity, and CYP2D6‐catalyzed Dextromethorphan dealkylation with IC50 values of 39, 42.5, and 57.5 μM, respectively. This in‐vitro data indicates that more in vivo studies should be conducted to examine the potential pharmacokinetic drug‐drug interactions with 9‐a due to its potent and moderate inhibition of CYP2A6, CYP2C9, CYP2E1, and CYP3A4.Inhibitory effect of WES‐1 on the major CYP enzymes in human liver microsomesFigure 1Inhibitory effect of 9a on the major CYP enzymes in human liver microsomesFigure 2

An in-vitro cocktail assay for assessing compound-mediated inhibition of six major cytochrome P450 enzymes

BackgroundProtein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy.Methods and FindingsEncouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors.ConclusionsOur results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.

Objectives Recent studies have demonstrated that inhibition of epidermal growth factor receptor (EGFR) kinase activity rapidly reduces tumor glucose metabolism in sensitive cell lines. This suggests that imaging of tumor metabolism with positron emission tomography (PET) with the glucose analog [ 18 F]fluorodeoxyglucose (FDG) could be used clinically to monitor treatment with EGFR kinase inhibitors. In this study, I investigated whether EGFR kinase inhibition specifically affects glucose metabolism or whether it also affects lipid and amino acid metabolism. In doing so, I am attempting to determine the best PET tracers to identify malignancies responding to EGFR kinase inhibitors in the clinics. Methods The lung cancer cell lines H3255 and A549 served as models for tumors that are sensitive and resistant to EGFR kinase inhibition, respectively. Glucose, amino acid, and lipid metabolism were studied by measuring the uptake of radiolabeled analogs of glucose, tyrosine, and choline. Uptake of these tracers was assayed in vitro after incubating cells with increasing doses of the EGFR kinase inhibitor erlotinib and in vivo by PET imaging of tumor bearing mice before and after erlotinib treatment. In vitro blocking studies with specific reagents were performed to elucidate the mechanisms of tyrosine and choline uptake. Results In sensitive H3255 cells, erlotinib decreased glucose uptake 54 ± 2% within 4 hours, whereas tyrosine and choline uptake were unaffected. In resistant A549 cells, no reduction of tracer uptake was observed for any of the studied tracers. These in vitro findings were confirmed in H3255 xenografted mice, which demonstrated a selective reduction of glucose uptake by 50 ± 5% after two doses of erlotinib. In A549 xenografts, erlotinib had no measurable metabolic effects. In H3255 and A549 cells, 2-amino-2-norbornanecarboxylic acid, a specific inhibitor of l-type amino acid transport, reduced tyrosine uptake by 83 ± 1% and 75 ± 1%, and cold choline inhibited choline uptake with IC50s of 5 μM and 6 μM, respectively. Conclusion These observations suggest that efficacious erlotinib treatment specifically modulates glucose, but not amino acid or lipid metabolism, as EGFR kinase inhibition may not affect the l-type amino acid transport system or the high-affinity choline transport system. Thus, the glucose analog FDG is the most promising tracer for monitoring treatment with EGFR kinase inhibitors in the clinics.

Pancreatic cancer is an extremely lethal malignancy whose prognosis has not improved appreciably in the last decade. The disease is usually diagnosed only after it has reached an advanced or metastatic state but even local, surgically resectable, tumors have a fairly poor prognosis. This is further complicated by the fact that little is known about the underlying mechanisms of pancreatic cancer, as evidenced by a lack of any efficacious targeted therapies. While hyperactive epidermal growth factor receptor (EGFR), Src tyrosine kinase and Signal Transducer and Activator of Transcription (Stat)3 have been detected in pancreatic cancer and implicated in the disease, the exact mechanisms by which they promote the disease phenotype are not well understood. We present evidence of a functional heteromeric complex of EGFR, c-Src and Stat3 that is detected at the plasma membrane and cytoplasm, and inside the nucleus of pancreatic cancer cells. The inhibition of c-Src, but not EGFR kinase, reduced the level of nuclear EGFR levels. Furthermore, the inhibition of Src or EGFR kinase alone decreased nuclear Stat3 levels, while the greatest decrease occurred upon the combined inhibition of EGFR and Src kinase activities. Significantly, evidence shows that the nuclear EGFR, Src and Stat3 complex functions as a transcriptional complex. Thus, the siRNA knockdown of EGFR or Src, or the pharmacological inhibition of aberrant Stat3 activity only moderately decreased c-Myc, iNOS, and VEGF expression. By contrast, the knockdown of EGFR or Src together with the inhibition of Stat3 activity strongly suppressed c-Myc expression in Panc-1 and Colo-357 cells. Chromatin immunoprecipitation (ChIP) studies further validated these findings and showed that the nuclear EGFR, Src, and Stat3 complex is bound to the c-Myc promoter. Taken together, these data identify a novel and functional heteromeric EGFR, Src, and Stat3 complex that promotes the induction of c-Myc in pancreatic cancer cells. Our study provides additional insight into the mechanisms by which hyperactive EGFR, Src and Stat3 pathway promotes the pancreatic cancer phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2031. doi:10.1158/1538-7445.AM2011-2031

The clinical evidence for the success of tyrosine kinase inhibitors in combination with microtubule-targeting agents prompted us to design and develop single agents that possess both epidermal growth factor receptor (EGFR) kinase and tubulin polymerization inhibitory properties. A series of 6-aryl/heteroaryl-4-(3',4',5'-trimethoxyanilino)thieno[3,2- d]pyrimidine derivatives were discovered as novel dual tubulin polymerization and EGFR kinase inhibitors. The 4-(3',4',5'-trimethoxyanilino)-6-( p-tolyl)thieno[3,2- d]pyrimidine derivative 6g was the most potent compound of the series as an antiproliferative agent, with half-maximal inhibitory concentration (IC50) values in the single- or double-digit nanomolar range. Compound 6g bound to tubulin in the colchicine site and inhibited tubulin assembly with an IC50 value of 0.71 μM, and 6g inhibited EGFR activity with an IC50 value of 30 nM. Our data suggested that the excellent in vitro and in vivo profile of 6g may be derived from its dual inhibition of tubulin polymerization and EGFR kinase.

Pyrazoline-linked carboxamide derivatives were designed, synthesized, and evaluated for potential epidermal growth factor receptor (EGFR) kinase inhibition, anticancer activity, and apoptotic and cardiomyopathy toxicity. Compounds 6m and 6n inhibit EGFR kinase at a concentration of 6.5 ± 2.91 and 3.65 ± 0.54 µM, respectively. Some of these compounds showed effects on proliferation, which were also then evaluated against four different human cancer cell lines, that is, MCF-7 (breast cancer), A549 (non-small-cell lung tumor), HCT-116 (colon cancer), and SiHa cells (cancerous tissues of the cervix uteri). The results showedthat certainsynthetic compounds showed significant inhibitor activity; compounds 6m and 6n were more cytotoxic than doxorubicin against A549 cancer cells, with IC50 values of 10.3 ± 1.07 and 4.6 ± 0.57 µM, respectively. Additionally, compounds 6m and 6n induced apoptosis in A549 cancer cells, as evidenced by 4',6-diamidino-2-phenylindole (DAPI) staining and phase-contrast microscopy. Potency to induce apoptosis by compound 6n was further confirmed by fluorescence-activated cell sorting using Annexin V-FITC and propidium iodide labeling. Compound 6n showed normal cardiomyocytes with no marked sign of pyknotic nuclei in cardiomyopathy and also normal histological appearance of the renal cortex when compared with that of control. Results of molecular docking studies suggested that compounds 6m and 6n can bind to the hinge region of the adenosine triphosphate-binding site of EGFR kinase, like the standard drug erlotinib. Therefore, the present study suggests that compounds 6m and 6n have potent in vitro antitumor activities against the human non-small-cell lung tumor cell line A549, which can be further explored in other cancer cell lines and in animal studies.

Introduction: Focal Adhesion Kinase (FAK) is a critical signaling molecule showing elevated expression in nearly all tumor types. Phosphorylation of key tyrosine residues within FAK underlies its dual roles as both a kinase and a molecular scaffold that integrates signals from major oncogenes. Though FAK has emerged as a major drug target, inhibitors developed to date singularly target its kinase activity, leaving the scaffolding of oncogenic drivers intact. Our studies demonstrate that cells treated with FAK kinase inhibitors PF-573228, PF-562271, and defactinib retain phosphorylation of several key tyrosine residues downstream of oncogenic receptor tyrosine kinases (RTKs). Methods: We examined the phosphorylation status of key FAK scaffolding sites Y861 and Y925 in RTK expressing normal and cancer cells treated with FAK kinase inhibitors PF-573228, PF-562271, and defactinib. As Src kinase is known to directly phosphorylate Y861 and Y925, we also tested phosphorylation of these sites by RTKs in Src/Yes/Fyn (SYF) MEFs, which lack the major Src family kinases. Results: In cancer cells treated with FAK kinase inhibitors, multiple RTKs (HER2/3, PDGFR, and EGFR) activated FAK through the phosphorylation of effector tyrosines. Activation of HER2/3 in MDA-MB-453 breast cancer cells, PDGFR in U-87 glioblastoma cells, and EGFR in A549 lung cancer cells all stimulated Y861/Y925 phosphorylation in the presence of each of the three FAK kinase inhibitors. Moreover, Y861/Y925 phosphorylation occurred downstream of PDGFR and EGFR in Src-null SYF MEFs, demonstrating that RTK FAK targeting does not require Src. Finally, in vitro studies demonstrated that RTKs can directly phosphorylate these sites on FAK. Conclusion: We have identified a novel mechanism through which FAK retains signaling activity in the presence of kinase inhibitors. Continued FAK signaling in tumor cells which overexpress HER2, EGFR or PDGFR may therefore be a mode of clinical resistance to FAK kinase inhibitors. Citation Format: Sheila Figel, Felicia Lenzo, Timothy Marlowe, William Cance. Oncogenic drivers reactivate kinase-inhibited focal adhesion kinase (FAK) through phosphorylation of effector residues. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C77.

Patients with breast cancer do not respond to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in the clinic, although 56% of basal breast cancers overexpress EGFR. These observations suggest that inhibiting EGFR kinase activity is insufficient to abrogate tumor growth. Therefore, the contribution of EGFR overexpression in breast cancer remains unknown. Recently, the increased activation of other tyrosine kinases, such as Met and c-Src, has been shown to contribute to cellular responses to EGFR inhibitors. The non-receptor tyrosine kinase c-Src is overexpressed in 70% of breast cancers, yet like EGFR, its role in breast cancer tumorigenesis has not fully been elucidated. Using cell culture models, co-overexpression of EGFR and c-Src has been shown to enhance proliferation, transformation, and tumorigenesis. In addition, we have shown that inhibiting EGFR and c-Src kinase activities in combination results in a synergistic decrease in cell viability. Therefore, EGFR cooperativity with other kinases may mediate continued tumor growth and viability in breast cancer. The role of EGFR and c-Src co-overexpression in breast cancer metastasis remains unknown. Metastasis is a multi-step process that involves migration of tumor cells away from the primary tumor site. Here we present data demonstrating that when EGFR and c-Src are co-overexpressed in breast cancer cells (either endogenously or by inducible overexpression) there is an increase in EGF driven chemotactic cell migration. Interestingly, this EGF-induced increase in cell migration is dependent on the kinase activity of EGFR and c-Src, such that inhibition of either EGFR or c-Src kinase activity abrogates EGF-induced migration. This is in contrast to viability assays using the same cell lines where inhibiting EGFR kinase activity is insufficient to abrogate viability in EGFR/c-Src co-expressing cells. EGFR and c-Src crosstalk has been shown to result in the phosphorylation of EGFR on tyrosine 845. Tyrosine 845 on EGFR is not an autophosphorylation site and unlike other tyrosine residues found in the activation loops of tyrosine kinases, tyrosine 845 phosphorylation is not required for kinase activity, although it is required for EGFR/c-Src induced proliferation. In the EGFR and c-Src co-overexpressing breast cancer cell lines, treatment with either EGFR or c-Src kinase inhibitors led to a reduction in the phosphorylation on EGFR at tyrosine 845, correlating with the loss of EGF-mediated migration. In addition, mutation of tyrosine 845 on EGFR to a phenylalanine inhibited EGF-induced cell migration. These data suggest a role for Y845-EGFR phosphorylation in EGF-induced cell migration in EGFR/c-Src co-overexpressing breast cancer cells. In summary, we demonstrate that inhibiting either EGFR or c-Src kinase activity may be an effective way reduced breast cancer cell migration and abrogate metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2911. doi:10.1158/1538-7445.AM2011-2911

The epidermal growth factor receptor (EGFR) is over-expressed in approximately 90% of non-small cell lung cancer (NSCLC) and as such, blockade of EGFR activity has been a primary therapeutic target for NSCLC. As patients with wild-type EGFR have demonstrated only modest benefit from EGFR tyrosine kinase inhibitors (TKIs) there is a need for additional therapeutic approaches in patients with wild-type EGFR. The extracellular matrix (ECM) has been shown to play an important role in tumor growth and response to therapy, and focal adhesion kinase (FAK) expression, a key component of signaling downstream of ECM binding to cell surface integrins, has been shown to correlate with aggressive stage in NSCLC. As the FAK-Src signaling axis can activate EGFR signaling independently of EGFR ligand-binding and kinase activity, the use of FAK tyrosine kinase inhibitors in combination with EGFR TKIs was assessed as a means of enhancing response to treatment in NSCLC. Treatment of EGFR TKI-resistant NSCLC cells (A549 and H1299 with wild-type EGFR, and H1975 with T790M acquired resistance mutation in EGFR) with FAK tyrosine kinase inhibitor PF-573,228 alone decreased cell viability. Treatment of these cell lines with a combination of PF-573,228 and the EGFR TKI erlotinib was more effective at reducing cell viability and cell migration than either treatment alone. Additionally, the growth of EGFR TKI-resistant NSCLC cells in 3-dimensional culture was significantly impaired with a combination of FAK inhibitor and erlotinib compared to either treatment alone. Interestingly, although erlotinib alone could inhibit the phosphorylation of Akt to an extent in NSCLC cell lines, the combination of erlotinib and FAK inhibitor was able to almost completely inhibit Akt phosphorylation. As persistent Akt activity is associated with lack of response to EGFR TKIs, the enhanced reduction in cell viability seen with the addition of a FAK inhibitor to treatment with erlotinib appears to be, at least in part, due to enhanced inhibition of Akt activity in these cells. The efficacy of the combination treatment was confirmed in vivo using a xenograft model with subcutaneously implanted A549 cells in nude mice. Significant inhibition of tumor growth was observed for A549-derived tumors when erlotinib was used in combination with FAK inhibitor, with some animals not yet developing palpable tumors by the end-point of the experiment. Thus, the combination of FAK inhibition with EGFR TKIs such as erlotinib results in decreased growth of NSCLC cells both in vitro and in vivo and could prove to be an effective therapeutic approach for patients with EGFR TKI-resistant NSCLC. Citation Format: Grant A. Howe, Bin Xiao, Huijun Zhao, Glenwood Goss, Christina L. Addison. Enhanced anti-tumor activity of erlotinib in combination with FAK tyrosine kinase inhibitors in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 753. doi:10.1158/1538-7445.AM2014-753

The epidermal growth factor (EGF) receptor is a tyrosine kinase that dimerizes in response to ligand binding. Ligand-induced dimerization of the extracellular domain of the receptor promotes formation of an asymmetric dimer of the intracellular kinase domains, leading to stimulation of the tyrosine kinase activity of the receptor. We recently monitored ligand-promoted conformational changes within the EGF receptor in real time using luciferase fragment complementation imaging and showed that there was significant movement of the C-terminal tail of the EGF receptor following EGF binding (Yang, K. S., Ilagan, M. X. G., Piwnica-Worms, D., and Pike, L. J. (2009) J. Biol. Chem. 284, 7474-7482). To investigate the structural basis for this conformational change, we analyzed the effect of several mutations on the kinase activity and luciferase fragment complementation activity of the EGF receptor. Mutation of Asp-960 and Glu-961, two residues at the beginning of the C-terminal tail, to alanine resulted in a marked enhancement of EGF-stimulated kinase activity as well as enhanced downstream signaling. The side chain of Asp-960 interacts with that of Ser-787. Mutation of Ser-787 to Phe, which precludes this interaction, also leads to enhanced receptor kinase activity. Our data are consistent with the hypothesis that Asp-960/Glu-961 represents a hinge or fulcrum for the movement of the C-terminal tail of the EGF receptor. Mutation of these residues destabilizes this hinge, facilitating the formation of the activating asymmetric dimer and leading to enhanced receptor signaling.

The focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) are protein-tyrosine kinases that are overexpressed and activated in human breast cancer. To determine the role of EGFR and FAK survival signaling in breast cancer, EGFR was stably overexpressed in BT474 breast cancer cells, and each signaling pathway was specifically targeted for inhibition. FAK and EGFR constitutively co-immunoprecipitated in EGFR-overexpressing BT474 cells. In low EGFR-expressing BT474-pcDNA3 vector control cells, inhibition of FAK by the FAK C-terminal domain caused detachment and apoptosis via pathways involving activation of caspase-3 and -8, cleavage of poly(ADP-ribose) polymerase, and caspase-3-dependent degradation of AKT. This apoptosis could be rescued by the dominant-negative Fas-associated death domain, indicating involvement of the death receptor pathway. EGFR overexpression did not inhibit detachment induced by the FAK C-terminal domain, but did suppress apoptosis, activating AKT and ERK1/2 survival pathways and inhibiting cleavage of FAK, caspase-3 and -8, and poly(ADP-ribose) polymerase. Furthermore, this protective effect of EGFR signaling was reversed by EGFR kinase inhibition with AG1478. In addition, inhibition of FAK and EGFR in another breast cancer cell line (BT20) endogenously overexpressing these kinases also induced apoptosis via the same mechanism as in the EGFR-overexpressing BT474 cells. The results of this study indicate that dual inhibition of FAK and EGFR signaling pathways can cooperatively enhance apoptosis in breast cancers.