Factor Receptor Tyrosine Kinase Activity Research Articles

Unexpected Need of the Epidermal Growth Factor Receptor Tyrosine Kinase Activity for Signaling by Intracellular Pattern Recognition Receptors of Nucleic Acids.

Many pattern recognition receptors in mammalian cells initiate signaling processes that culminate in mounting an innate protective response mediated by induced synthesis of a large number of proteins including type I interferons and other cytokines. Many of these receptors are not located on the plasma membrane but on the membranes of intracellular organelles such as endosomes, mitochondria, and the endoplasmic reticulum; they primarily recognize microbial or cellular nucleic acids. In the course of biochemical analyses of the signaling pathways triggered by these receptors, we discovered that they require tyrosine phosphorylation by the protein kinase activity of the epidermal growth factor receptor (EGFR), which is located not only on the plasma membrane but also on the intracellular membranes. Here, we discuss how specific members of this family of receptors, such as TLR3, TLR9, or STING, interact with EGFR and other protein tyrosine kinases and what are the functional consequences of their post-translational modifications. The article highlights an unexpected functional link between a growth factor receptor and cellular innate immune response.

Synthesis, structural characterization, and molecular docking studies of bioactive bismuth(III) complexes with substituted hydrazones

Eight new bismuth(III) complexes (1-8) of substituted hydrazones having general formula; [Bi(RCONHNCHC5H4N)Clx] and [Bi(RCONHNCHC9H6N)Clx], where R = C10H7O (1, and 8), C4H3S (2), C6H5O (3, and 6), C7H7(4), C5H4N (5, and 7), x = 2 or 3 have been prepared. The substituted hydrazones (I1-I8) were synthesized by reacting the stoichiometric amounts of the respective hydrazides such as p-toluic hydrazide, 4-hydroxybenzhydrazide, thiophene-2-carboxylic acid hydrazide, 3-hydroxy-2-naphthoic acid hydrazide, Isonicotic acid hydrazide and appropriate aromatic aldehydes like pridine-2-carboxaldehyde and quinoline-2-carboxyaldehyde. These hydrazones (ligands),then complexed to Bi(III) species to yield the target compounds. They were characterized by FTIR, and NMR spectroscopy to find out an explicit evidence about their structural motifs. The X-ray data for (1 & I6) further validate the chemical structures of the synthesized compounds. The molecular geometry for (1) is predominately distorted pentagonal bipyramidal where hepta-coordinated Bi(III) center is attached to the nitrogens of pyridine and of azomethine moieties along with the carbonyl oxygen, two chloro and two oxygens of the (two) DMSO molecules. The bond lengths for Bi(1)-O(1), Bi(1)-N(1) and Bi(1)-N(2) are 2.323(5), 2.539(6), 2.390(5) oA respectively indicating Bi(1)-O(1) as the strongest linkage to the metal center. The H-bonding, present in (1), contributed an extra stability to the structure. The synthesized compounds were initially screened for their possible antimicrobial, alpha-amylase, and protein kinase inhibition, some (1,2,6,8) exhibit significant activity. The experimental results are further endorsed by molecular docking studies against helicobacter pylori urease (1E9Y), epidermal growth factor receptor tyrosine kinase (1M17) and human pancreatic alpha amylase (5U3A) inhibition activity. The significant ligand-receptor interactions, observed at the binding pocket of the selected drug targets, further revealed the enzyme inhibition potential of the compounds and highlighting their possible roles as therapeutic agents in future drug discovery processes.

The Biological Role of Hyaluronan-Rich Oocyte-Cumulus Extracellular Matrix in Female Reproduction.

Fertilization of the mammalian oocyte requires interactions between spermatozoa and expanded cumulus extracellular matrix (ECM) that surrounds the oocyte. This review focuses on key molecules that play an important role in the formation of the cumulus ECM, generated by the oocyte-cumulus complex. In particular, the specific inhibitors (AG1478, lapatinib, indomethacin and MG132) and progesterone receptor antagonist (RU486) exerting their effects through the remodeling of the ECM of the cumulus cells surrounding the oocyte have been described. After gonadotropin stimulus, cumulus cells expand and form hyaluronan (HA)-rich cumulus ECM. In pigs, the proper structure of the cumulus ECM depends on the interaction between HA and serum-derived proteins of the inter-alpha-trypsin inhibitor (IαI) protein family. We have demonstrated the synthesis of HA by cumulus cells, and the presence of the IαI, tumor necrosis factor-alpha-induced protein 6 and pentraxin 3 in expanding oocyte-cumulus complexes (OCC). We have evaluated the covalent linkage of heavy chains of IαI proteins to HA, as the principal component of the expanded HA-rich cumulus ECM, in porcine OCC cultured in medium with specific inhibitors: AG1478 and lapatinib (both inhibitors of epidermal growth factor receptor tyrosine kinase activity); MG132 (a specific proteasomal inhibitor), indomethacin (cyclooxygenase inhibitor); and progesterone receptor antagonist (RU486). We have found that both RU486 and indomethacin does not disrupt the formation of the covalent linkage between the heavy chains of IαI to HA in the expanded OCC. In contrast, the inhibitors AG1478 and lapatinib prevent gonadotropin-induced cumulus expansion. Finally, the formation of oocyte-cumulus ECM relying on the covalent transfer of heavy chains of IαI molecules to HA has been inhibited in the presence of MG132.

The activation of SRC family kinases and focal adhesion kinase with the loss of the amplified, mutated EGFR gene contributes to the resistance to afatinib, erlotinib and osimertinib in human lung cancer cells.

Second- and third-generation inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase activity (EGFR-TKIs) are improving the treatment of patients with non-small cell lung cancer. Here we established two sublines (BR1-8 and BR2-3) resistant to a second-generation inhibitor, afatinib, from the human lung cancer cell line HCC827 that harbors a mutation that activates the tyrosine kinase activity of EGFR. These afatinib-resistant sublines were resistant to first-generation EGFR-TKIs, gefitinib and erlotinib, and a third-generation EGFR-TKI, osimertinib. These resistant sublines showed markedly reduced levels of multiple EGFR family proteins, including the activated mutant EGFR, and complete loss of EGFR amplification as compared with their parental HCC827 cells harboring amplification of EGFR gene. Treatment with the multikinase inhibitor dasatinib or transfection with a SRC small interfering RNA inhibited cell survival and AKT phosphorylation in drug-resistant sublines to a greater extent compared with HCC827 cells. Further, the migration of drug-resistant cells was greater compared with that of HCC827 cells and was inhibited by dasatinib or an FAK inhibitor. These findings indicate that compensatory activation of SRC family kinases (SFKs) and FAK supports the survival and migration of afatinib-resistant cells when the expression of multiple EGFR family proteins was mostly abrogated. Combinations of potent drugs that target SFKs and FAK may overcome the resistance of lung cancer cells to second-generation TKIs.

Phosphorylation and Internalization of Lysophosphatidic Acid Receptors LPA1, LPA2, and LPA3.

ResultsThe lysophosphatidic acid receptors LPA1, LPA2, and LPA3 were individually expressed in C9 cells and their signaling and regulation were studied. Agonist-activation increases intracellular calcium concentration in a concentration-dependent fashion. Phorbol myristate acetate markedly inhibited LPA1- and LPA3-mediated effect, whereas that mediated by LPA2 was only partially diminished; the actions of the phorbol ester were inhibited by bisindolylmaleimide I and by overnight incubation with the protein kinase C activator, which leads to down regulation of this protein kinase. Homologous desensitization was also observed for the three LPA receptors studied, with that of LPA2 receptors being consistently of lesser magnitude; neither inhibition nor down-regulation of protein kinase C exerted any effect on homologous desensitization. Activation of LPA1–3 receptors induced ERK 1/2 phosphorylation; this effect was markedly attenuated by inhibition of epidermal growth factor receptor tyrosine kinase activity, suggesting growth factor receptor transactivation in this effect. Lysophosphatidic acid and phorbol myristate acetate were able to induce LPA1–3 phosphorylation, in time- and concentration-dependent fashions. It was also clearly observed that agonists and protein kinase C activation induced internalization of these receptors. Phosphorylation of the LPA2 subtype required larger concentrations of these agents and its internalization was less intense than that of the other subtypes.ConclusionOur data show that these three LPA receptors are phosphoproteins whose phosphorylation state is modulated by agonist-stimulation and protein kinase C-activation and that differences in regulation and cellular localization exist, among the subtypes.

Icotinib hydrochloride enhances the effect of radiotherapy by affecting DNA repair in colorectal cancer cells.

The aim of the present study was to explore the efficacy and mechanism of the radiosensitisation of icotinib hydrochloride (IH), a novel oral epidermal growth factor receptor-tyrosine kinase activity inhibitor, by evaluating the changes in tumour cell double-strand breaks (DSBs) repair, cell cycle and apoptosis following a combination of IH and radiotherapy (RT) in human colorectal adenocarcinoma cell lines. The HT29 and HCT116 human CRC cell lines were treated with IH and/or radiation. Effects on cell viability and cell cycle progression were measured by MTT, a clonogenic survival assay, and flow cytometry. Immunofluorescent staining and western blot analysis were applied to detect the expression of γ-H2AX and 53BP1 in the different treatment groups. Finally, the in vivo effect on the growth of CRC xenografts was assessed in athymic nude mice. IH inhibited the proliferation and enhanced the radiosensitivity in HT29 and HCT116 CRC cells lines. IH combined with radiation increased cell cycle arrest in the G2/M phase compared to the other treatments in the HT29 cell line (P<0.05). Similarly, cell cycle arrest occurred in the HCT116 cell line, although this increase did not result in significant differences in the RT group (P>0.05). IH combined with radiation significantly inhibited the expression of γ-H2AX and 53BP1 based on results of immunofluorescent staining and western blot analysis. In vivo, IH plus radiation significantly inhibited the tumour growth compared to either agent independently. In conclusion, IH significantly increased the radiosensitivity of HT29 and HCT116 cells in vitro and in vivo. Radiation combined with EGFR blockade inhibited tumour proliferation, increased apoptosis, prolonged G2/M arrest and significantly enhanced DNA injury in colorectal cancer. These data support the clinical trials of biologically targeted and conventional therapies in the treatment of cancer.

Anti-epidermal growth factor receptor tyrosine kinase activities of traditional Chinese medicine for cancer treatment

IntroductionThe epidermal growth factor receptor (EGFR) is a validated target for different human malignancies. EGFR tyrosine kinase inhibitors considerably extend progression-free survival in EGFR-mutant non-small cell lung cancer. Monoclonal antibodies targeting EGFR also improve the efficacy outcomes in KRAS wild-type colorectal cancer. At present, gefitinib and erlotinib are considered the most representative of these types of drugs approved by the FDA for cancer treatment. However, only a few studies have screened the active compounds of traditional Chinese medicines. MethodsIn this study, the EGFR inhibitory activities of Chinese medicinal herb extracts traditionally prescribed to treat cancer were determined in a 384-well plate-based assay using the homogeneous time-resolved fluoroimmunoassay method. Petroleum ether, ethanol, ethyl acetate, and aqueous extracts of 49 traditional Chinese medicinal herbs (183 extracts total) were tested. The inhibitory effects were expressed as percentage of inhibition. Dose-dependent inhibitory assays were also performed for the herbal extracts that exhibited significant inhibition. ResultsThe ethyl acetate extracts of Artemisia argyi, Lonicera macranthoides, Spatholobus suberectus, Curcuma longa, and Galla chinensis, as well as the ethanol extract of Eriobotrya japonica exhibited significant EGFR inhibitory activities at a concentration of 50μg/ml. The IC50 values were 8.848, 2.027, 2.277, 3.621, 4.339 and 5.528μg/mL, respectively. The EA extracts of L. macranthoides, S. suberectus had the best activities. ConclusionOur results indicate that screening traditional Chinese herbs for EGFR inhibitory activity may identify a large number of potential lead compounds for the development of new drugs to treat cancer.

Pro-MMP-9 upregulation in HT1080 cells expressing CD9 is regulated by epidermal growth factor receptor

Degradation of the surrounding extracellular matrix (ECM) by matrix metalloproteinases (MMPs) drives invasion and metastasis of cancer cells. We previously demonstrated that tetraspanin CD9 expression upregulates pro-MMP-9 expression and release and promotes cellular invasion in a human fibrosarcoma cell line (HT1080). These events were dependent upon the highly functional second extracellular loop of CD9. We report here that the epidermal growth factor receptor (EGFR) tyrosine kinase expression and activity are involved in the CD9-mediated increase in pro-MMP-9 release and cellular invasion. Pro-MMP-9 expression was significantly decreased in a dose-dependent manner using first a broad spectrum receptor tyrosine kinase inhibitor and multiple specific EGFR inhibitors in CD9-HT1080 cells. Furthermore, gefitinib treatment of CD9-HT1080 cells reduced invasion through matrigel. EGFR knockdown using short interfering RNA resulted in decreased pro-MMP-9 expression and release into the media and subsequent cellular invasion without affecting CD9 expression or localization. Conclusively, this study points to EGFR as a key mediator between CD9-mediated pro-MMP-9 release and cellular invasion of HT1080 cells.

Erlotinib-induced Adverse Skin Reactions

Erlotinib is low molecular-weight quinazolin derivatives which selectively inhibit the epidermal growth factor receptor (EGF-R) tyrosine kinase activity of the intracellular domain, block autophosphorylation and the subsequent signaling cascades. EGF-R is expressed on basal keratinocytes, sebocytes, the outer root sheath of hairs, and endothelial cells in the skin, and plays important roles in the regulation of differentiation, proliferation, apoptosis, attachment and migration of keratinocytes, inflammation, and wound healing. Therefore, inhibition of EGF-R causes a number of cutaneous adverse reactions. Among them, severe skin lesions are very stressful, and impair quality of life of patients. Moreover, they even bring disadvantages such as drug withdrawal or interruption. Several review papers describe representative or common skin lesions which appear either during the first a few weeks or at later phases. Common skin manifestations include papular and pustular follicular eruptions (acneiform eruption), xerosis, paronychia, pruritus, and abnormalities of hairs; however, other than those eruptions, several unusual lesions are also induced. Early intervention of dermatologists and management of skin lesions are quite important, because discontinuance of the drug is unfavorable for patients with clinical benefits for cancers. In this brief review, various cutaneous manifestations seen in Japanese patients treated with erlotinib (Tarceva) are shown, and current management of representative severe conditions is also described.

Tyrosine Phosphorylation of Mig6 Reduces Its Inhibition of the Epidermal Growth Factor Receptor

Under physiological conditions, epidermal growth factor receptor (EGFR) tyrosine kinase activity is tightly controlled through the coordinated action of both positive and negative regulators. Aberrant EGFR activation occurs frequently in many cancer types, and the endogenous EGFR feedback inhibitor, Mig6/RALT, is more efficiently phosphorylated by oncogenic EGFR variants. We have utilized expressed protein ligation to generate semisynthetic Tyr394 phosphorylated and unphosphorylated forms of the Mig6 protein and shown that phosphorylation of Mig6 reduces its ability to inhibit purified, near full-length EGFR (tEGFR). We also demonstrate that the kinetic parameters of tEGFR are similar whether solubilized in detergent or reconstitutued in nanodisc bilayers. These findings suggest a mechanism by which EGFR and its family members evade negative regulation by Mig6 under pathological conditions.

Effects of broth culture filtrate protein of VacA+ Helicobacter pylori on the proliferation and apoptosis of gastric epithelial cells

Background Infection with Helicobacter pylori (H. pylori) may lead to chronic inflammation of the stomach epithelium, mucosal atrophy, imbalance of proliferation and apoptosis of epithelial cells; resulting in chronic gastritis, peptic ulcer, gastric cancer, and many other clinical outcomes. Why and how H. pylorus leads to gastric cancer is not clear yet. Through in vitro experiments, this study evaluated the effects of broth culture filtrate protein (BCF-P) from the supernatant of liquid culture media of H. pylori on proliferation and apoptosis of immortalized human gastric epithelial cell lines (GES-1) and gastric cancer cell lines (AGS). Methods For the study, GES-1 and AGS cell lines mix with BCF-P and epidermal growth factor (EGF). MTT assay and flow cytometry (FCM) determined the levels of proliferation and apoptosis. Detected expression levels of cyclooxygenase-2 (COX-2) and Fas mRNA by reverse transcription (RT)-PCR. Also did analysis of the effects of BCF-P on epidermal growth factor receptor (EGFR) tyrosine kinase activity of GES-1 and AGS cells by non-radioactive enzyme-linked assay. The Student’s t test and one-way analysis of variance (ANOVA) were used for statistical analysis. Results BCF-P inhibited proliferation of GES-1 and AGS cells in a concentration-dependent manner. The inhibition rates are respectively 68.7% in AGS and 61.4% in GES-1. With the same dose and time for inhibiting the proliferation, BCF-P failed to induce apoptosis of GES-1 and AGS cells. Effects of BCF-P reduced the expression of Fas mRNA of GES-1 and AGS cells (P &lt;0.05). This is consistent with the effects of EGF. BCF-P reduced the expression of COX-2 mRNA of AGS cells (P &lt;0.05). This is opposite to the effects of EGF (P &lt;0.05). Effects of BCF-P improved more than three times the EGFR tyrosine kinase activity of GES-1 and AGS cells. Conclusions BCF-P inhibited the proliferation of AGS and GES-1 cells in vitro, unrelated to apoptosis. Effects of BCF-P on gastric epithelial cells in vitro are not equivalent to that of EGF.

Inhibition of platelet-derived growth factor receptor tyrosine kinase and downstream signaling pathways by Compound C

AMP-activated protein kinase (AMPK) has been implicated in anti-proliferative actions in a range of cell systems. Recently, it was observed that Compound C, an inhibitor of AMPK, also reduced the cell viability in human diploid fibroblasts (HDFs). Compound C-induced growth arrest was associated with a decrease in the cell cycle regulatory proteins, such as proliferating cell nuclear antigen, phosphorylated pRB, cyclin-dependent protein kinases (Cdk 2 and 4), cyclins (D and E), and the Cdk inhibitors (p21, p16, and p27). Therefore, the present study examined the molecular mechanism of the antiproliferative effects of Compound C. Although Compound C inhibited serum-induced phosphorylation of Akt and its substrate, glycogen synthase kinase-3β, it did not affect the Akt activity in vitro. Compound C significantly inhibited the receptor tyrosine phosphorylation and the activity of downstream signaling molecules, such as p85 phosphoinositide 3-kinase, phospholipase C-γ1, and extracellular signal-regulated kinase 1/2, induced by platelet-derived growth factor (PDGF) but not by epidermal growth factor- and insulin-like growth factor. In vitro growth factor receptor tyrosine kinase activity profiling revealed the IC50 for PDGF receptor-β (PDGFRβ) to be 5.07μM, whereas the IC50 for the epidermal growth factor receptor and insulin-like growth factor receptor was ≥100μM. The inhibitory effect of Compound C on PDGFRβ and Akt was also observed in AMPKα1/α2-knockout mouse embryonic fibroblasts, indicating that its inhibitory effect is independent of the AMPK activity. The inhibitory effect of Compound C on cell proliferation and PDGFRβ tyrosine phosphorylation was also demonstrated in various PDGFR-expressing cells, including MRC-5, BEAS-2B, rat aortic vascular smooth muscle cells, and A172 glioblastoma cells. These results indicate that Compound C can be used as a potential antiproliferative agent for PDGF- or PDGFR-associated diseases, such as cancer, atherosclerosis, and fibrosis.

Abstract 5232: Tyrosine kinase inhibitor, gefitinib targets ZAP-70 over expressing chronic lymphocytic leukemia cells

Abstract Most patients with chronic lymphocytic leukemia (CLL) initially respond to chemotherapy but relapse. There is a subset of CLL patients that have aggressive disease characterized by an over expression of the tyrosine kinase ZAP-70. These ZAP-70+ patients have a poorer survival and are more resistant to chemotherapy. The biological effects of ZAP-70 in CLL are believed to be related to its ability to enhance activation of Syk with subsequent triggering of down-stream modulators, such as ERK and AKT. Normally, Syk is activated by increased phosphorylation by Lyn, which is increased in CLL, or by activation of the B-cell receptor. Gefitinib is a tyrosine kinase inhibitor that is presently used to treat lung cancer. The drug inhibits epidermal growth factor receptor (EGFR) tyrosine kinase activity but has activity against &amp;gt;20 other kinase targets, including members of the Src family of kinases, such as Lyn and Syk. We thus evaluated the cytotoxic activity of gefitinib against primary CLL cells using the MTT assay. Gefitinib was cytotoxic to ZAP-70+ (≥20% cells positive) CLL samples (median IC50, ∼3.0 µM) while ZAP-70- patients failed to respond to gefitinib with a median IC50 of &amp;gt;15.0 µM. Jurkat cells, which are T cells that express high levels of ZAP-70, were more sensitive to gefitinib than B cell lines that did not express ZAP-70 (BJAB and NALM6). Further studies confirmed that gefitinib was inducing cell death through apoptosis. Gefitinib was also effective against ZAP-70+ CLL cells that were resistant to fludarabine and chlorambucil indicating a novel mechanism of action to standard chemotherapy. Enhanced apoptosis was observed in ZAP70+ CLL and Jurkat cells exposed to gefitinib and fludarabine or chlorambucil, but this was not observed in ZAP-70- or the B cell lines. In contrast, another EGFR inhibitor, erlotinib, had no effect against ZAP-70+ CLL cells indicating that gefitinib was inhibiting a kinase unaffected by erlotinib. When Jurakat and ZAP-70+ CLL cells were treated with gefitinib, there was reduced total tyrosine phosphorylation and decreased tyrosine phosphorylation of ZAP-70, and Syk but no difference in Lyn phosphorylation. Furthermore, gefitinib blocked B cell receptor activation and mediated cell survival. Taken together, these results indicate that gefitinib may be useful agent in the treatment of ZAP-70+ CLL, either alone or in combination with fludarabine or chlorambucil. Ongoing studies are determining the precise mechanism of action of gefitinib in ZAP-70+ CLL and assessing its effects on the CLL microenvironment. 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 5232. doi:1538-7445.AM2012-5232

Synthesis and biological evaluation of 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-substituted-phenoxy)pyrimidines as dual EGFR/ErbB-2 kinase inhibitors

A series of 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-substituted-phenoxy)pyrimidine derivatives were elaborately designed based on the skeleton of Lapatinib, and evaluated for their potential to inhibit epidermal growth factor receptor (EGFR) and ErbB-2 tyrosine kinase activities and antiproliferative activities against A431 and SKOV-3 cell lines. Among these synthesized pyrimidine derivatives, 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-acrylamidophenoxy)pyrimidine (6), 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-cyanoacetamidophenoxy)pyrimidine (9), 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-{3-[6-(4-amino)pyrimidinyl]amino) phenoxy}pyrimidine (11) and 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-phenoxyacetamidophenoxy)pyrimidine (14) could significantly inhibit dual EGFR/ErbB-2 kinase activities (IC50=37/29nM, 48/38nM, 61/42nM, 65/79nM, respectively). And compounds 6 and 11 also showed the most potent antiproliferative activities in vitro, with the IC50 value of 6 being 3.25μM for A431 and 0.89μM for SKOV-3, as for 11, 4.24μM for A431 and 0.71μM for SKOV-3, respectively. Docking study was also performed to determine the possible binding model.

Diffuse Interstitial Lung Disease Linked to Vandetanib

Vandetanib inhibits vascular endothelial growth factor receptor (VEGFR) 2 tyrosine kinase activity in endothelial cells and, to a lesser degree, endothelial growth factor receptor (EGFR) tyrosine kinase activity in tumor cells. Vandetanib has been developed for the treatment of non–small-cell lung cancer (NSCLC), either alone or in combination with second-line chemotherapy. We describe a case of interstitial lung disease (ILD) in a patient who received vandetanib in a randomized, controlled, double-blind trial by comparing a pemetrexed combination with either vandetanib or placebo.

Abstract 2032: Regulation of MUC1 expression by activated EGFR and PPARγ in human uterine and pancreatic cancer cell lines

Abstract Many epithelial tumors including endometrial and pancreatic cancers, overexpress the high molecular weight, transmembrane mucin, MUC1, on their cell surface. Overexpression and aberrant glycosylation of MUC1 makes tumor cells poorly adherent, increases drug resistance and promotes cancer cell metastasis. MUC1 has three distinct domains: an extracellular domain (ECD) composed of a tandem repeat region rich in serine, threonine and proline; a transmembrane domain (TM) and; a short cytoplasmic tail domain (CT) involved in a variety of signal transduction events. Previous studies (Wang et al. 2010. Mol. Endocrinol. 24: 1368-1379) demonstrated that the Peroxisome Proliferator Activated Receptor γ (PPARγ) agonist, rosiglitazone severely inhibits progesterone stimulated MUC1 expression. Two independent approaches were used to determine if rosiglitazone as well as inhibitors of Epidermal Growth Factor Receptor (EGFR) tyrosine kinase activity reduced MUC1 expression in the human uterine epithelial cell line, HES, the uterine adenocarcinoma cell line, HEC-1A, and in the human pancreatic adenocarcinoma cell lines, HPAFII and CAPAN2. EGFR activation by Epidermal Growth Factor (EGF) stimulated (1.5-2 fold) MUC1 protein expression 1.5-2.0 fold in both uterine and pancreatic cancer cells, while treatment with EGFR tyrosine kinase inhibitor, AG1478, reduced both basal and stimulated MUC1 protein expression. Furthermore, the PPARγ agonist, rosiglitatzone, markedly (&amp;gt; 90%) reduced EGFR-driven MUC1 expression detected by antibodies directed against the MUC1 ECD and CT. A combination of biochemical and molecular biological assays have been used to identify the transcriptional and signal transduction pathways activated by EGFR leading to increased MUC1 expression and the impact of AG1478 and rosiglitazone on these events. Understanding the molecular basis of how these agents reduce MUC1 expression may offer novel therapeutic avenues to improve cancer chemotherapies. 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 2032. doi:10.1158/1538-7445.AM2011-2032

Drug localization in different lung cancer phenotypes by MALDI mass spectrometry imaging

Lung cancer is a common cause of cancer mortality in the world, largely due to the risk factor of tobacco smoking. The drug therapy at the molecular level includes targeting the epidermal growth factor receptor (EGFR) tyrosine kinase activity by using inhibitors, such as erlotinib (Tarceva) and gefitinib (Iressa). The heterogeneity of disease phenotypes and the somatic mutations presented in patient populations have a great impact on the efficacy of treatments using targeted personalized medicine. In this study, we report on basic physical and chemical properties of erlotinib and gefitinib in three different lung cancer tumor phenotypes, using MALDI instrumentation in imaging mode, providing spatial localization of drugs without chemical labeling. Erlotinib and gefitinib were analyzed in i) planocellular lung carcinoma, ii) adenocarcinoma and iii) large cell lung carcinoma following their deposition on the tissue surfaces by piezo-dispensing, using a controlled procedure. The importance of high-resolution sampling was crucial in order to accurately localize the EGFR tyrosine kinase inhibitors deposited in heterogeneous cancer tissue compartments. This is the first report on personalized drug characterization with localizations at a lateral resolution of 30μm, which allowed us to map these compounds at attomolar concentrations within the lung tumor tissue microenvironments.

Evaluation of radioiodinated quinazoline derivative as a new ligand for EGF receptor tyrosine kinase activity using SPECT

A radioiodinated analog of PD153035 (m-IPQ) was evaluated as a potential epidermal growth factor receptor tyrosine kinase (EGFR-TK) activity imaging ligand for SPECT. The 50% inhibition concentration (IC₅₀) value of m-IPQ for EGFR-TK phosphorylation inhibition was evaluated and compared to various EGFR-TK inhibitors. [(125)I]m-IPQ was synthesized by iododestannylation reaction. Biodistribution study of [(125)I]m-IPQ was conducted in normal mice and tumor-bearing mice. The selectivity and binding characteristics (B(max) and K(d)) were analyzed. The quinazoline derivative m-IPQ was found to have high inhibitory potency (IC₅₀: 50.5 ± 3.5 nM) and selectivity toward EGFR-TK. In vivo biodistribution studies of [(125)I]m-IPQ demonstrated its rapid clearance and low retention in normal tissue. On the other hand, high tumor uptake was observed. However, the increase in [(125)I]m-IPQ uptake in the stomach as a deiodination parameter was found. Thus, [(125)I]m-IPQ showed low in vivo stability. The selectivity toward EGFR-TK of m-IPQ was confirmed by the pretreatment experiment with EGFR-TK specific inhibitors, PD153035, Genistein. [(125)I]m-IPQ bound to single population of binding sites with high affinity and kinetic parameter. In addition, [(125)I]m-IPQ was bound to EGFR-TK according to the amount of EGFR-TK expression in the tumor. [(125)I]m-IPQ showed a relatively high tumor accumulation with selective EGFR-TK binding. Moreover, the tumor uptake of [(125)I]m-IPQ might be reflected in the amount of EGFR-TK expression in the tumor. These good characteristics of [(125)I]m-IPQ suggested that a ¹²³I-labeled counterpart, [¹²³I]m-IPQ, would have great potential for EGFR-TK imaging with SPECT. However, the in vivo stability of this compound needs to improve.

Hypoxia-induced proliferation of human pulmonary microvascular endothelial cells depends on epidermal growth factor receptor tyrosine kinase activation

We hypothesized that hypoxia would activate epidermal growth factor receptor (EGFR) tyrosine kinase, leading to increased arginase expression and resulting in proliferation of human pulmonary microvascular endothelial cell (hPMVEC). To test this hypothesis, hPMVEC were incubated in normoxia (20% O(2), 5% CO(2)) or hypoxia (1% O(2), 5% CO(2)). Immunoblotting for EGFR and proliferating cell nuclear antigen was done, and protein levels of both total EGFR and proliferating cell nuclear antigen were greater in hypoxic hPMVEC than in normoxic hPMVEC. Furthermore, hypoxic hPMVEC had greater levels of EGFR activity than did normoxic hPMVEC. Hypoxic hPMVEC had a twofold greater level of proliferation compared with normoxic controls, and this increase in proliferation was prevented by the addition of AG-1478 (a pharmacological inhibitor of EGFR). Immunoblotting for arginase I and arginase II demonstrated a threefold induction in arginase II protein levels in hypoxia, with little change in arginase I protein levels. The hypoxic induction of arginase II protein was prevented by treatment with AG-1478. Proliferation assays were performed in the presence of arginase inhibitors, and hypoxia-induced proliferation was also prevented by arginase inhibition. Finally, treatment with an EGFR small interfering RNA prevented hypoxia-induced proliferation and urea production. These findings demonstrate that hypoxia activates EGFR tyrosine kinase, leading to arginase expression and thereby promoting proliferation in hPMVEC.

UTP Controls Cell Surface Distribution and Vasomotor Activity of the Human P2Y2 Receptor through an Epidermal Growth Factor Receptor-transregulated Mechanism

Extracellular nucleotides transmit signals into the cells through the P2 family of cell surface receptors. These receptors are amply expressed in human blood vessels and participate in vascular tone control; however, their signaling mechanisms remain unknown. Here we show that in smooth muscle cells of isolated human chorionic arteries, the activation of the P2Y(2) receptor (P2Y(2)R) induces not only its partition into membrane rafts but also its rapid internalization. Cholesterol depletion with methyl-beta-cyclodextrin reduced the association of the agonist-activated receptor into membrane rafts but did not affect either the UTP-mediated vasoconstrictions or the vasomotor responses elicited by both serotonin and KCl. Ex vivo perfusion of human chorionic artery segments with 1-10 mum UTP, a selective P2Y(2)R agonist, displaced the P2Y(2)R localization into membrane rafts within 1 min, a process preceded by the activation of both RhoA and Rac1 GTPases. AG1478, a selective and potent inhibitor of the epidermal growth factor receptor tyrosine kinase activity, not only blocked the UTP-induced vasomotor activity but also abrogated both RhoA and Rac1 activation, the P2Y(2)R association with membrane rafts, and its internalization. Altogether, these results show for the first time that the plasma membrane distribution of the P2Y(2)R is transregulated by the epidermal growth factor receptor, revealing an unsuspected functional interplay that controls both the membrane distribution and the vasomotor activity of the P2Y(2)R in intact human blood vessels.