Receptor Tyrosine Kinases EGFR Research Articles

Abstract 2509: AV-203, a humanized ERBB3 inhibitory antibody inhibits ligand-dependent and ligand-independent ERBB3 signaling in vitro and in vivo

Abstract ERBB3 is a member of the EGFR receptor tyrosine kinase (RTK) family. Agents targeting the family of EGFR RTKs have become widely used for the treatment of lung, colon, breast, gastric, and head and neck cancers. Among EGFR family members, ERBB3 is of special interest because of its ability to activate the survival pathway driven by PI3K, its essential role in HER2 mediated cancers, and its involvement in tumor progression and drug resistance. The ERBB3 receptor is expressed on cells from cancers of the head and neck, lung, breast, ovaries, prostate, colon, pancreas, and gastrointestinal tract. Its expression is often linked to poor prognosis. In addition, it has been implicated in the development of resistance to current anti-cancer agents including receptor-targeted tyrosine kinase inhibitors (TKI). ERBB3 lacks detectable tyrosine kinase activity and its activation requires the heterodimerization of ERBB3 with RTK partners, i.e., HER2, EGFR or MET. ERBB3 recruitment in heterodimer complexes takes place when RTKs are overexpressed or amplified or stimulated by ligands, i.e. Neuregulin-1 (NRG1) or epidermal growth factor (EGF). Because of the lack of ERBB3 kinase activity, antibody therapies directed against the extracellular domain of ERBB3 seem to be the most effective method to disrupt the function of ERBB3. Herein, characterization of AV-203, a humanized ERBB3 inhibitory antibody, is presented. AV-203 is a humanized immunoglobulin G1/kappa antibody that targets the ERBB3 RTK. AV-203 binds to human ERBB3 with high affinity (KD = 76 pM at 37°C) characterized by fast association and slow dissociation rates. AV-203 also binds to cynomolgus monkey ERBB3 but not to mouse ERBB3, allowing toxicological assessment of the antibody in this species. AV-203 is a potent inhibitor of ERBB3 activation and its downstream signaling molecule AKT in response to both ligands, NRG1 and EGF. In ligand-independent settings, AV-203 inhibits the steady state activation of ERBB3/AKT which depends on the presence of an overexpressed RTK such as HER2. AV-203 can prevent the ERBB3/HER2 heterodimer formation and completely inhibit the proliferation in response to NRG1 ligand in human breast cancer cell line, MCF7. AV-203 down regulates ERBB3 receptor in vitro and in vivo. Finally, AV-203 inhibits tumor growth in a broad spectrum of xenograft models in which ERRB3 is activated by its ligand NRG1 or by HER2 overexpression such as the pancreatic cancer BxPC3 or the HER2 amplified breast cancer MDA-MB-453 xenograft models, respectively. In conclusion, AV-203 demonstrated high affinity binding to ERBB3, potent inhibition of NRG1 binding and of ERBB3 activation in ligand-dependent and ligand-independent manners both in vitro and in vivo. First in human trial of AV-203 in cancer patients is planned for 2012. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2509. doi:1538-7445.AM2012-2509

Steroid-like signalling by interferons: making sense of specific gene activation by cytokines

Many cytokines, hormones and growth factors use the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathway for cell signalling and specific gene activation. In the classical model, ligand is said to interact solely with the receptor extracellular domain, which triggers JAK activation of STATs at the receptor cytoplasmic domain. Activated STATs are then said to carry out nuclear events of specific gene activation. Given the limited number of STATs (seven) and the activation of the same STATs by cytokines with different functions, the mechanism of the specificity of their signalling is not obvious. Focusing on IFNγ (interferon γ), we have shown that ligand, receptor and activated JAKs are involved in nuclear events that are associated with specific gene activation, where the receptor subunit IFNGR1 (IFNγ receptor 1) functions as a transcription/co-transcription factor and the JAKs are involved in key epigenetic events. RTKs (receptor tyrosine kinases) such as EGFR [EGF (epidermal growth factor) receptor] and FGFR [FGF (fibroblast growth factor) receptor] also undergo nuclear translocation in association with their respective ligands. EGFR and FGFR, like IFNGR1, have been shown to function as transcription/co-transcription factors. The RTKs also regulate other kinases that have epigenetic effects. Our IFNγ model, as well as the RTKs EGFR and FGFR, have similarities to that of steroid receptor signalling. These systems consist of ligand-receptor-co-activator complexes at the genes that they activate. The co-activators consist of transcription factors and kinases, of which the latter play an important role in the associated epigenetics. It is our view that signalling by cytokines such as IFNγ is but a variation of specific gene activation by steroid hormones.

Emerging Systemic Therapeutic Approaches for Personalized Medicine in Squamous Cell Carcinoma of the Lung

During the past decade, a number of novel agents developed for the treatment of advanced NSCLC had, by coincidence, shown survival benefit predominantly among patients with nonsquamous histology, namely pemetrexed (anti-folate), bevacizumab (VEGF pathway inhibition), erlotinib/gefitinib (EGFR receptor tyrosine kinase inhibitors) and crizotinib (ALK receptor tyrosine kinase inhibitor. This has sparked a heightened interest in discovering key molecular aberrations that may be relevant for the treatment of squamous cell carcinoma (SCC) of the lung, a histologic subtype that represents approximately 25% of lung cancer cases diagnosed globally. This article highlights the most promising recent discoveries and targeted agents in development that may be relevant for the treatment of SCC of the lung.

Abstract LB-212: XGFR, an Fc-engineered dual signaling inhibitor targeting IGF-1R and EGFR

Abstract Background: Elevated signaling via the receptor tyrosine kinases IGF-1R and EGFR has been identified as common characteristic of multiple cancer type. IGF-1R and EGFR signal predominantly through the PI3K and MAPK signaling pathways and thereby mediate growth and survival signals crucial for the development and progression of cancer. There is strong cross talk on multiple levels between IGF-1R and EGFR dependent signaling pathways. Therefore, targeting IGF-1R and EGFR simultaneously is an attractive way to achieve maximal inhibition of signal transduction and to avoid resistance formation. Methods: Bispecific IGF1R-EGFR antibodies were engineered by linking scFv domains of an EGFR Mab (GA201) via Serine-Glycine linkers to an IgG1 IGF-1R Mab (RG1507). The functional properties of the bispecific antibodies were evaluated in cellular in vitro assays (IGF-1R/EGFR phosphorylation, downregulation, 3D proliferation and ADCC assays) and in in vivo xenograft models for tumor growth inhibition and survival. Results: Bispecific IGF-1R-EGFR antibodies (XGFR2, XGFR3, XGFR4) were successfully generated with yields and stability comparable to conventional IgG1 antibodies. XGFR antibodies effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation in H322M tumor cells and induced strong downmodulation of IGF-1R and enhanced EGFR downmodulation compared to the parental EGFR antibody GA201. XGFR antibodies showed strong anti-tumor efficacy comparable to the combination of monospecific IGF-1R and EGFR Mabs in the BxPC3 and H322M xenograft models. To enhance the ADCC properties of XGFR, afucosylated, glycoengineered bispecific antibodies with enhanced affinity for FcγRIIIA were generated using the GlycoMab technology. Glycoengineered bispecific antibodies were shown to have superior ADCC properties in in vitro ADCC assays and XGFR4 significantly prolonged median and overall survival of mice in an ADCC competent in vivo model (A549 i.v.). Conclusions: Bispecific IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components of multiple cancer types (IGF-1R and EGFR), resulting in effective inhibition of the PI3K and MAPK signaling pathway and to avoid the formation of resistance to therapy. Having overcome issues of stability and productivity, bispecific antibodies may become an advantageous way to reduce costs and infusion times in cancer therapy, while at the same time, achieving maximal anti-tumor effects through inhibition of multiple targets. 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 LB-212. doi:10.1158/1538-7445.AM2011-LB-212

Epidermal growth factor enhances the malignant phenotype in canine mammary carcinoma cell lines

Canine mammary gland tumours (CMTs) are the most common malignancies in female dogs. The receptor tyrosine kinase EGFR (erbb1), a receptor for epidermal growth factor (EGF) and related factors, mediates multiple oncogenic functions in human epithelial neoplasms. While previous studies have demonstrated EGFR expression in canine tumours, its function has not been studied in canine cancer. The purpose of this study was to determine the in vitro effects of EGF and vandetanib (ZD6474), a small molecule inhibitor of VEGFR-2, EGFR and RET tyrosine kinases, on proliferation, invasion, survival and chemosensitivity in CMT cells. In low serum, EGF enhanced proliferation and chemotaxis, attenuated apoptosis, and stimulated vascular endothelial growth factor (VEGF) production. Vandetanib dose-dependently inhibited EGFR phosphorylation as well as PI3K/Akt activation, and inhibited all EGF-induced phenotypic effects. In conclusion, EGF stimulates multiple features promoting the malignant phenotype in CMT. Thus, CMT may be an important translational model for the investigation of novel EGFR-directed therapies.

Abstract 5422: Drug combination studies in a panel of 30 cell lines established from patient-derived tumor xenograft

Abstract In recent years, the focus of anticancer drug development has changed from conventional cytotoxic drugs to targeted agents interfering with cell proliferation, migration or survival. In most cases, signaling pathways, key regulatory complexes or biological processes of pathophysiological relevance are selected and proteins with key functions targeted. These include signaling cascades originating from receptor tyrosine kinases (e.g. c-Met, EGFR or HER2), the mitotic machinery (eg5, Aurora / ARK or polo-like kinases / Plk), protein turnover (Hsp70/90, 26S proteasome) and the nucleosome (e.g. HDACs, HATs). However, as a single agent new targeted drugs often demonstrate weak antitumor activity in preclinical testing and clinical trials. Combinations of new targeted agents or with standard cytotoxic drugs are a suitable strategy resulting in potent anti-cancer therapies. As the number of possible drug combinations is essentially limitless and a scientific rationale is only available in few cases, a screening strategy to identify the most promising drug combinations in vitro is crucial for success in further development. At Oncotest a panel of 30 proprietary solid tumor cell lines established from patient-derived xenografts is routinely used. All major histologies are represented such as NSCLC, colon and breast cancer, as well as niche tumors like pleuramesothelioma, bladder and liver cancer. Chemosensitivity information for most standard-of-care drugs and experimental compounds as well as genomic and proteomic characterization data are available. By using this cell line panel, systematic combination studies were performed with the propidium iodide standard cytotoxicity / proliferation assay. Synergistic or antagonistic activity was assessed according to the method of Chou-Talalay by calculating “Combination Index” (CI) values using the CalcuSyn software. Activity profiles were established for well described experimental an approved agents targeting for example c-Met (PF-04217903), EGFR / HER2 (Lapatinib, Erlotinib), Hsp90 (17-DMAG), Eg5 (Ispinesib, Monastrolin), Plk1 (BI2536), ARK A/B (Tozasertib), HDAC (Vorinostat, Etinostat) in combination with each other as well as with standard chemotherapeutic agents like Cisplatin, Paclitaxel or 5-Fluorouracil. The most promising combinations will be presented, for example the combination of HDAC or c-met inhibitors with EGFR receptor tyrosine kinase inhibitors displaying additive or synergistic activity. In conclusion, the Oncotest solid tumor cell line panel is suitable for a broad, systematic evaluation of drug combinations including cytotoxic and new targeted anti-cancer agents to identify potential beneficial combinations for further preclinical and clinical studies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5422.

Abstract 3625: Exogenous short-term activation of c-Met and EGFR sensitizes them to kinase inhibitors

Abstract Activation of the c-Met and EGFR receptor tyrosine kinases induces brain tumor malignancy and is associated with poor clinical outcome of brain tumor patients. Consequently, c-Met and EGFR are promising targets for the therapy of brain tumors and clinically applicable inhibitors of these receptors have been developed by biotechnology and pharmaceutical companies. We tested the effects of a specific and potent c-Met kinase inhibitor (PF-2341066) on glioma cells and glioma stem cells. Surprisingly, we found that the inhibitor induced significantly greater cell death when c-Met was shortly pre-activated with its ligand hepatocyte growth factor (HGF). Glioblastoma cells (U87, U373), primary glioblastoma cells (GBM10) and glioblastoma stem cells (1228, 0308) were pre-treated with 20 ng/ml HGF for 2h, 4h, 6h, 8h, 16h or 24h prior to treatment with PF-2341066 (100 nM for established and primary cells and 300 nM for cancer stem cells) for 24 hr. The cells were subsequently assessed for apoptosis using annexin-V flow cytometry. The results showed that the inhibitor induced significantly greater (34-79%) absolute cell death in pre-activated cells than in non-activated cells. The optimal time of pre-activation that achieved greatest sensitization of the receptor to kinase inhibitor-induced cell death was 2-4 hours. Using a similar experimental approach, we also tested the effects of EGFR pre-activation on EGFR inhibitor erlotinib-induced cell death in glioma cells. Glioma cells were pre-treated with EGF (100ng/ml) for 3h, 6h, 16h or 24h prior to treatment with erlotinib (2.5 µM) for 24 hrs and assessment of cell death by annexin V flow cytometry. EGFR pre-activation led to significantly greater (138 %) erlotinib-induced cell death than without pre-activation. Based on the above findings, we hypothesize that short-term exogenous activation of c-Met, EGFR and possibly also other receptor tyrosine kinases can trigger fast oncogene addiction that sensitizes tumor cells to death induced by receptor tyrosine kinase inhibitors. We are currently testing this hypothesis using in vivo glioblastoma animal models. Verification of this new hypothesis would imply that quick short-term activation of tyrosine kinase receptors by bolus delivery of receptor tyrosine kinase ligands would sensitize tumors to kinase inhibitors and therefore improve the efficacy of these inhibitors in cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3625.

Abstract 3228: Dual HER2 and EGFR inhibitor BIBW2992 have dramatic activity in a HER2 wild type/EGFR L858R murine lung cancer model

Abstract Dual HER2 and EGFR inhibitor BIBW2992 have dramatic activity in a HER2 wild type/EGFR L858R murine lung cancer model HER2 (ERBB2) is a proto-oncogene that belongs to EGFR tyrosine kinase receptor family. HER2 genomic locus amplification and HER2 protein over-expression detected by IHC have been identified in many human cancer types including breast, brain and lung cancer. Simultaneous over-expression of both HER2 and EGFR occurs in a subpopulation of lung cancer patients. Here we developed a transgenic mouse model with inducible wild type HER2 expression in type II pneumocyte. Continual HER2 over-expression in the lung epithelial compartment leads to peripheral lung adenocarcinoma that responds dramatically to targeted anti-HER2 therapeutics. Mice over-expressing both EGFR L858R and the wild type HER2 alleles do not significant accelerate tumorigenesis or alter of tumor histology as compared to tumors generated by the wild type HER2 single alleles. The HER2/EGFR driven murine lung tumors showed mild and transient response to the EGFR inhibitor, gefitinib. This result mimics the observation from clinical patients. However, the dual HER2/EGFR inhibitor, BIBW2992, is much more effective against these HER2/EGFR driven lung cancers as measured by tumor regression and over all survival. Our data suggested that targeting therapy against both EGFR and HER2 is a better strategy in lung cancer patients with co-expression of EGFR and HER2 when compared with treatment against EGFR alone. Clinical trial for dual HER2/EGFR inhibitors in this population should be considered. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3228.

Abstract 620: Addition of silibinin to EGFR-TKIs overcomes T790M-mediated drug resistance

Abstract Although epidermal growth factor receptor EGFR tyrosine kinase inhibitors (EGFR-TKIs) produce an initially exhibit an initial dramatic response in lung cancer patients harboring a mutation in the EGFR gene, development of acquired resistanceis almost inevitable. The secondary mutation of T790M is associated with approximately one- half of the cases of acquired resistance. In this study, we investigated whether addition of silibinin to gefitinib or erlotinib could overcome T790M-mediated drug resistance considering that silibinin has various anti-tumor effects including EGFR modulation. Silibinin selectively reduced the activity of the EGFR family (EGFR, HER2 and ErbB3) in lung cancer cells with EGFR mutations, but not in those harboring wild type. In primary and acquired resistant cells with T790M, addition of silibinin enhanced the ability of EGFR-TKIs to down-regulate EGFR signals and to inhibit cell growth. Similarly, the combination of silibinin and erlotinib effectively suppressed the tumor growth in erlotinib resistance-bearing PC-9 xenografts. The results indicate that In conclusion, the addition of silibinin to EGFR-TKIs is can be a promising strategy to overcome T790M-mediated drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 620.

Synthesis and Biological Evaluation of 4-Anilinoquinolines as Potent Inhibitors of Epidermal Growth Factor Receptor

The mutant receptor tyrosine kinase EGFR is a validated and therapeutically amenable target for genotypically selected lung cancer patients. Here we present the synthesis and biological evaluation of a series of 6- and 7-substituted 4-anilinoquinolines as potent type I inhibitors of clinically relevant mutant variants of EGFR. Quinolines 3a and 3e were found to be highly active kinase inhibitors in biochemical assays and were further investigated for their biological effect on EGFR-dependent Ba/F3 cells and non-small cell lung cancer (NSCLC) cell lines.

Abstract B144: NVP-BEZ235, a dual inhibitor of PI3K and mTOR, induces MAPK phosphorylation in HER2-positive cells

Abstract The PI3K pathway is highly deregulated in human cancer with the most common alterations being amplification of the receptor tyrosine kinases (RTKs) EGFR and HER2 and mutations in PI3K itself or inactivation of the tumor suppressor PTEN. Thus, the rationale exists to develop therapeutic agents targeting the PI3K/AKT pathway. Several small molecule inhibitors targeting PI3K, AKT and mTOR are now in clinical trials. However, these novel compounds may interfere with regulatory feedback loops. One such example involves the activation of AKT following rapamycin analogues treatment, such as everolimus, an allosteric mTOR inhibitor. Blocking mTOR signaling also alters the regulation of other signaling pathways, like the MAPK cascade. The aim of this study was to investigate the mechanism/s by which inhibition of the PI3K pathway leads to activation of MAPK kinase signaling in breast cancer cell lines and its relevance in tumor treatment. We have observed an increased MAPK phosphorylation in HER2 positive BT474, SKBR-3, MDA-361 and MCF-7HER2 cells when treated with NVP-BEZ235, a dual inhibitor of both mTOR and p110, the catalytic component of PI3K. The increase of P-MAPK was observed by western blot and correlated with hyperphosphorylation of several RTKs. PD325901, a MEK1/2 inhibitor, or lapatinib, a HER1/2 tyrosine kinase inhibitor, reverted the NVP-BEZ235-induced MAPK phosphorylation. On the other hand, NVP-AEW541, an IGFR-1 tyrosine kinase inhibitor, did not prevent NVP-BEZ235-dependent HER2 phosphorylation and downstream MAPK activation. Activation of MAPK upon administration of NVP-BEZ235 was also observed in vivo. We are currently investigating the possible involvement of known regulatory feedback loops as well as the individual RTK contribution. Activation of parallel pathways in a condition of PI3K inhibition may reduce potential benefits of these anti-cancer treatments. For this reason, we propose the combination of an anti-HER2 therapy together with PI3K inhibitors for the treatment of HER2 positive tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B144.

Reply to Dr. Peparini’s Letter to the Editor regarding the publication titled “Immunohistochemical detection of receptor tyrosine kinases c-kit, EGF-R and PDGF-R in colorectal adenocarcinomas”

Dear Editor, We enjoyed reading the comments of Peparini et al. [1] in response to our recent publication entitled “Immunohistochemical detection of receptor tyrosine kinases c-kit, EGFR and PDGF-R in colorectal adenocarcinomas” [2] as it leads interest to the combination of two promising directions of cancer research. It is pointed out by Peparini and colleagues that epidermal growth factor receptor (EGF-R) is able to induce epithelial–mesenchymal transition (EMT) as shown for breast cancer [3] and that this mechanism may play a crucial role in tumor progression and formation of metastases. Emerging evidence suggests an important participation of EMT as regarding to tumor invasiveness which is underlined by a recent series of reviews published on this topic [4, 5]. However, signaling pathways in EMT and the relationship to tyrosine kinases are still poorly understood. In our study, out of 257 samples of colorectal carcinoma, only 39 (15.2%) stained positive for the expression of EGF-R, showing no significant correlation with pathological parameters which is not concordant with many studies. The controversy of differing detection rates has been discussed in our original article. A possible explanation not mentioned in our article is the heterogeneity of tumors with varying expression patterns in the bulk tumor, lymph nodes, metastases, and the tumor invasion front, representing regions of interest for EMT. For our study, we used tissue arrays of representative areas of the primary colorectal tumor. We did not perform specific counterstaining of the core needle biopsy, e.g., for EMT markers, but focusing expression patterns on different topographic sites of the same carcinoma, as proposed in the Letter to the Editor, might clarify the relationship of EMT with the expression of EGF-R and explain the variety of published expression patterns for colorectal carcinomas. In our study, we reported a detection rate of c-kit in only 1.9% of all 263 tumor samples, confirming the rare expression of c-kit in colorectal carcinomas described in literature. The discussed possibility of a different c-kitpositive tumor entity or the recently described lack of specificity of several kinase inhibitors leading to therapeutic benefit in c-kit-negative colorectal carcinomas [6] are just two interesting theories. The expression of c-kit at the tumor invasion front and the relationship to EMT or the reverting mesenchymal–epithelial transition, a suggested by Peparini and colleagues, definitively represent possible explanations and need further studies for clarification. In summary, we completely agree with Peparini and coworkers that research in this direction might lead to a better understanding of neoplastic progression and novel therapeutic strategies.

Expression of receptors tyrosine kinase c-kit and EGF-R in colorectal adenocarcinomas: is there a relationship with epithelial–mesenchymal transition during tumor progression?

To the Editor:We read with great interest the contribution by Friederichsand colleagues regarding immunohistochemical detectionof the receptor tyrosine kinases c-kit, EGF-R, and PDGF-Rin colorectal adenocarcinomas [1]. The authors showedEGF-R expression in only 15.3% of cases; emphasized thatactivity of tyrosine kinase inhibitors does not necessarilycorrelate with the tyrosine kinase expression of the tumors;showed that there is no correlation between EGF-R incolorectal cancer, clinical response to anti-EGF-R anti-bodies, and outcome; and that pretreatment selection ofpatients cannot be based on immunohistochemical expres-sion of EGF-R.EGF has recently been identified as a novel inducer ofepithelial–mesenchymal transition (EMT) in human breastcancer [2]. EMT is a process of plasticity by whichepithelial cells lose contact with adjacent cells andbasement membranes, switch patterns of extracellularmatrix protein and intermediate filament expression, andtransdifferentiate into mesenchymal cells. EMT duringtumor progression allows tumor cells to infiltrate surround-ing tissue and to metastatize to distant sites. A key event inEMT is reduction of cell–cell adhesion by transcriptionalrepression of E-cadherin. Loss of E-cadherin expression hasbeen reported in several cancers, including advancedcolorectal carcinomas. EMT hallmarks include reductionof E-cadherin with concomitant production of N-cadherinand acquisition of mesenchymal markers such as vimentinand fibronectin [3, 4]. EMT may influence the response ofcertain tumors to EGF-R-targeted therapies. In nonsmallcell lung cancer cell lines, sensitivity to an EGF-R-targetedmonoclonal antibody (erlotinib) did not correlate with EGF-R levels, but was dependent on EMT status: non-responding nonsmall cell lung carcinomas showed lowlevels of E-cadherin and higher levels of mesenchymalmarkers [5, 6].In the study by Friederichs et al., positive EGF-Rstaining did not show significant correlation with patho-logical parameters, but higher expression of EGF-R inadvanced and metastatic stages in colorectal adenocarcino-mas was reported in other studies.Friederichs et al. concluded that c-kit expression is rarein colorectal carcinomas, and because of the small numberof cases, it is uncertain if c-kit-positive carcinomasrepresent a specific entity or in which way these carcinomasdiffer. The authors suggested that in colorectal cancerwithout c-kit kinase activation, treatment with recentlydeveloped and less selective tyrosine kinase inhibitors mayresult in inhibition of alternative or related tyrosine kinase

Suppression of the Kinase Activity of Receptor Tyrosine Kinases by Anthocyanin-Rich Mixtures Extracted from Bilberries and Grapes

Two standardized anthocyanin-rich mixtures were investigated for their ability to inhibit the receptor tyrosine kinases (RTKs) EGFR, ErbB2, ErbB3, VEGFR-2, and VEGFR-3. Both mixtures reduced the kinase activity of recombinant kinase domains of each RTK at concentrations <or=12.9 microg/mL, with preferential inhibition of VEGFR-2 and EGFR (<or=3.4 microg/mL). Similarly, ligand-induced autophosphorylation of these RTKs in human vulva carcinoma or porcine aortic endothelial cells was suppressed by both mixtures, with ErbB3 and VEGFR-3 being preferentially inhibited. Anthocyanin-rich extracts completely abrogated VEGFR-3 phosphorylation at concentrations of >or=50 microg/mL. These results indicate that anthocyanin-rich mixtures can inhibit RTKs with low specificity. The rank order of inhibitory efficacy against the tested RTKs in intact cells was VEGFR-3 >> VEGFR-2 > ErbB3 > EGFR > ErbB2. Considering the important role of RTKs in carcinogenesis, their inhibition by anthocyanin-rich mixtures suggests that they may serve as biomarkers of the pharmacological efficacy of anthocyanins in future chemoprevention experiments and in clinical intervention studies.

Epidermal growth factor receptor activation by diesel particles is mediated by tyrosine phosphatase inhibition

Exposure to particulate matter (PM) is associated with increased cardiopulmonary morbidity and mortality. Diesel exhaust particles (DEP) are a major component of ambient PM and may contribute to PM-induced pulmonary inflammation. Proinflammatory signaling is mediated by phosphorylation-dependent signaling pathways whose activation is opposed by the activity of protein tyrosine phosphatases (PTPases) which thereby function to maintain signaling quiescence. PTPases contain an invariant catalytic cysteine that is susceptible to electrophilic attack. DEP contain electrophilic oxy-organic compounds that may contribute to the oxidant effects of PM. Therefore, we hypothesized that exposure to DEP impairs PTPase activity allowing for unopposed basal kinase activity. Here we report that exposure to 30 μg/cm 2 DEP for 4 h induces differential activation of signaling in primary cultures of human airway epithelial cells (HAEC), a primary target cell in PM inhalation. In-gel kinase activity assay of HAEC exposed to DEPs of low (L-DEP), intermediate (I-DEP) or high (H-DEP) organic content showed differential activation of intracellular kinases. Exposure to these DEP also induced varying levels of phosphorylation of the receptor tyrosine kinase EGFR in a manner that requires EGFR kinase activity but does not involve receptor dimerization. We demonstrate that treatment with DEP results in an impairment of total and EGFR-directed PTPase activity in HAEC with a potency that is independent of the organic content of these particles. These data show that DEP-induced EGFR phosphorylation in HAEC is the result of a loss of PTPase activities which normally function to dephosphorylate EGFR in opposition to baseline EGFR kinase activity.

Synergism of EGFR and c-Met pathways, cross-talk and inhibition, in non-small cell lung cancer

Backgroundc-Met and EGFR receptors are widely expressed on cancer cells; they are implicated in the development and progression of cancer through a plethora of effects on cell cycle progression, apoptosis, motility and metastasis and are potential targets for combination therapy. EGFR receptor tyrosine kinases are currently being targeted in a number of malignancies.MethodsApoptosis was studied by FACS analysis using propidium iodide. EGF and HGF signaling intermediates were studied by western blotting. Cell proliferation was determined by MTT assays. Cell motility was done by time lapse confocal microscopy.Resultsc-Met and EGFR were both expressed in A549, H1838, H2170, SW900, SW1573, H358, SKLU-1, and H1993 non small cell lung cancer (NSCLC) cell lines. Both EGF and HGF at 100 ng/ml in medium showed a synergistic effect on cell proliferation at 48–72 h as seen by a proliferation assay in A549, H1838, and SKMES cells. In A549 and H1838 cell lines, HGF (40 ng/ml) and EGF (5 ng/ml) induced synergistic phosphorylation on c-Met (Tyr 1003/1230/1234/1235). Additionally, synergistic phosphorylation of Akt (Ser-473) and phospho-ERK1+ERK2 (Thr202/Tyr204) was also seen indicating that EGF and HGF could induce synergistic phosphorylation of important signaling intermediates. Treatment with EGF and HGF at 100 ng/ml for 2 h also leads to an additive effect in inducing cell motility (especially membrane ruffling) in H1993 cells. A novel c-Met small molecule tyrosine kinase inhibitor SU11274 and EGFR tyrosine kinase inhibitors Tyrphostin AG1478 and gefitinib (Iressa) were tested to study their effect in combination on proliferation and apoptosis in lung cancer cells. Interestingly, a synergistic effect on inhibition of cell proliferation was seen in the presence of SU11274 and Tyrphostin AG1478. 0.5 µM Tyrphostin AG1478 and 2 µM SU11274 inhibited growth by 21% and 25%, respectively; a combination of both tyrosine kinase inhibitors inhibited growth by 65%. Interestingly, EGFR inhibitor (gefitinib, Iressa) and c-Met inhibitor (SU11274) also had a synergistic effect on apoptosis in H358 cells.ConclusionThere was a synergistic effect of EGF and HGF on proliferation, downstream activation of signal transduction and an additive effect seen on motility. These studies show that a combination of HGF and EGF tyrosine kinase inhibitors on NSCLC, could potentially be targeted in a synergistic fashion.

A phase I trial of gefitinib (ZD1839) plus rapamycin for patients with recurrent malignant glioma

1565 Abnormal signaling of the PI3K/Akt pathway is a hallmark of malignant glioma. The primary objective of this phase 1 study is to determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of Gefitinib, an inhibitor of the EGFR receptor tyrosine kinase, when combined with rapamycin, a macrolide antibiotic capable of inhibiting mTOR, a critical downstream regulator of PI3K/Akt signaling, among patients with recurrent malignant glioma. Eligibility criteria include: recurrent malignant glioma; no more than 3 prior episodes of recurrence; age greater than 18 years; KPS greater than 60%; adequate hepatic, renal, pulmonary and bone marrow function. Patients with prior with EGFR or mTOR-directed therapies are excluded. A standard “3+3” dose escalation design was employed. Patients are stratified based on concurrent use of enzyme-inducing anticonvulsants (EIAC; phenytoin, carbamazepine and phenobarbitol) and both strata are independently escalated. Gefitinib and rapamycin are administered on a continuous daily dosing regimen. Each treatment cycle is 28 days and evaluation for response is performed after every other cycle. Twenty-three patients have been enrolled to date: 20 with recurrent GBM and 3 with recurrent AA. The median age is 51 (range 33 to 66); 65% are male and 52% are on EIAC. Accrual and dose escalation are ongoing. The MTD has yet to be defined for either stratum. A single DLT has been observed thus far and consisted of an episode of grade 3 mucositis. Approximately half of the patients have undergone pharmacokinetic sampling. Seventeen patients continue on study having received 1–3 cycles of therapy while 6 patients have discontinued therapy due to progressive disease. Two marked radiographic responses has been observed to date. An update of the outcome and toxicity of Gefitinib plus rapamycin for patients with recurrent malignant glioma will be presented based on additional follow up and enrollment. No significant financial relationships to disclose.

Effects of prolonged tamoxifen treatment on receptor expression and apoptosis of ovarian cancer cells

Objective Tamoxifen, which is widely used in the treatment of breast cancer, also has a beneficial effect on cisplatin-refractory ovarian cancer. In this study, we investigated the long-term effects of this drug on estrogen-receptor-positive ovarian cancer cells. Methods We performed an in vitro selection process by long-term treatment of BG-1 ovarian cancer cells with 4-hydroxy tamoxifen (4-OH TAM). Drug effects on cell growth were determined by measurement of relative cell numbers (MTS assay), the apoptotic effects of 4-OH TAM were determined by analysis of poly (ADP-ribose) polymerase (PARP) cleavage and by ELISA measurement of DNA–histone complexes in cytoplasm. Results Analysis of BG-1(LT) ovarian cancer cells isolated after 5 months of long-term treatment with 4-OH TAM revealed both a significantly reduced apoptotic and antiproliferative effect of this drug. Further experiments to examine expression changes of the receptor tyrosine kinases EGFR, HER2 and estrogen receptor α did not reveal any alterations in BG-1(LT) if compared to wild-type cells. In contrast, in this cell line, a significant alteration in the expression of estrogen receptor β was observed. Conclusion Our findings indicate that long-term treatment with 4-OH TAM is able to diminish both the antiproliferative and apoptotic action of this drug on BG-1 ovarian cancer cells. Our data suggest that the responsiveness of ovarian cancer cells to 4-OH TAM decreases after long-term treatment with this drug in vitro like previously observed after long-term treatment of breast cancer cells.

The effect of combination treatment with gemcitabine and the EGFR-receptor tyrosine kinase inhibitor iressa with photodynamic therapy in bladder cancer

The effect of combination treatment with gemcitabine and the EGFR-receptor tyrosine kinase inhibitor iressa with photodynamic therapy in bladder cancer

Signaling by G protein coupled receptors and G proteins: a perspective

Cell surface receptors belonging to the family of G protein coupled receptors (GPCRs) ± also known as seven-transmembrane, heptahelical or serpentine, receptors ± are involved in the regulation of diverse signaling processes. Heterotrimeric G proteins, consisting of a, b, and g-subunits provide the signal coupling mechanisms to this superfamily of receptors. The role of the heptahelical receptors and their G proteins in signaling mechanisms involving sensory transduction as well as cell homeostasis has been known for almost three decades. However, only in the recent days, their critical role in the regulation of cell growth has been identi®ed. The identi®cation of newer heptahelical receptors as well as G protein subunits has contributed towards the greater understanding of the novel mechanisms through which these receptors recruit G proteins and small GTPases in regulating cell growth. The compendium of reviews presented here focuses on the mechanisms by which the di€erent components of the heptahelical receptors signaling unit, namely the receptors, the G protein subunits, and their e€ectors regulate the critical signaling events involved in cell growth, di€erentiation, and apoptosis. In the reviews presented here, the authors bring their unique perspective to describe the possible mechanisms by which signaling pathways regulated by G protein coupled receptors and G proteins contribute to signal networking involved in cell growth, di€erentiation and apoptosis. Signaling by GPCRs is initiated by a speci®c ligand b that binds and activates the receptor inducing a conformational change in the receptor. The receptor, thus activated, stimulates a speci®c G protein by catalyzing the exchange of guanine nucleotides in the a-subunit. The GTP-bound a-subunit and the ba subunits disassociate from the receptor and activate their respective down-stream e€ector proteins. The ®rst six articles from this compendium analyse the signaling events from the vantage point of the GPCRs. The following two articles describe the variations of the common theme exhibited by two novel families of GPCRs namely, the PAR and the virally encoded GPCRs. Two articles that discuss the intracellular communication networking initiated by GPCRs involving receptor tyrosine kinases, non-receptor tyrosine kinases, and STATs follow this. As the signal from GPCR progresses to the G proteins, the next series of seven reviews discusses the mechanisms by which di€erent G proteins transduce the signals from the receptors to the downstream e€ector molecules. The review by Lefkowitz and coworkers describe the novel mechanisms by which GPCRs activate the extracellular-signal regulated kinases (ERKs) and Jun N-terminal kinases (JNK) through the recruitment of receptoras well as non-receptor associated kinases. In the article by Kraneburg and Moolenaar, the mechanism by which lysophosphatidic receptors selectively coupling to speci®c G proteins to recruit the small GTPase Ras in activating the ERK signaling pathway. In contrast, the role of Rho-family of GTPases in cellular transformation induced by the novel GPCRs are described by Channing Der and his colleagues. The role of such GPCRs in angiogenesis and the possible role of this pathway in metastasis are discussed by Jacques Pouysse gur and his co-workers. In the next article, Lynn Heasley reviews the autocrine and paracrine signaling pathways stimulated by neuropeptide receptors and their possible role in human cancers. In the following article, Lawrence Brass and coworkers summarize novel mechanisms by which the PAR-family of GPCRs are activated. Recent studies have shown that some human pathogenic viruses utilize either endogenous or virally encoded GPCRs for their growth and survival pathways. Schwartz and his colleagues discuss the novel aspects of these virally encoded GPCRs. Critical signaling pathways involved in cell proliferation, di€erentiation, and apoptosis is regulated by multiple signaling input. More often than not, these multiple signaling inputs are generated from signaling pathways originating from di€erent signaling loci. A potent transforming, di€erentiating, or apoptotic signal involves the activation of these multiple signaling pathways. The article by Axel Ullrich and his colleagues describe the mechanism through which GPCRs can transactivate the receptor tyrosine kinase EGFR to promote cell proliferation. Similarly the article by Ram and Iyengar describe the mechanism(s) by which GPCRs especially the receptors that couple Oncogene (2001) 20, 1530 ± 1531 a 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00