Epidermal growth factor receptor mutation in combination with expression of MIG6 alters gefitinib sensitivity

<p>Abstract</p> <p>Background</p> <p>Epidermal growth factor receptor (EGFR) signaling plays an important role in the regulation of cell proliferation, survival, metastasis, and invasion in various tumors. Earlier studies showed that the EGFR is frequently overexpressed in non-small-cell lung cancer (NSCLC) and EGFR mutations at specific amino acid residues in the kinase domain induce altered responsiveness to gefitinib, a small molecule EGFR tyrosine kinase inhibitor. However, the mechanism underlying the drug response modulated by EGFR mutation is still largely unknown. To elucidate drug response in EGFR signal transduction pathway in which complex dynamics of multiple molecules involved, a systematic approach is necessary. In this paper, we performed experimental and computational analyses to clarify the underlying mechanism of EGFR signaling and cell-specific gefitinib responsiveness in three H1299-derived NSCLC cell lines; H1299 wild type (H1299WT), H1299 with an overexpressed wild type EGFR (H1299EGFR-WT), and H1299 with an overexpressed mutant EGFR L858R (H1299L858R; gefitinib sensitive mutant).</p> <p>Results</p> <p>We predicted and experimentally verified that Mig6, which is a known negative regulator of EGFR and specifically expressed in H1299L858R cells, synergized with gefitinib to suppress cellular growth. Computational analyses indicated that this inhibitory effect is amplified at the phosphorylation/dephosphorylation steps of MEK and ERK.</p> <p>Conclusions</p> <p>Thus, we showed that L858R receptor mutation in combination with expression of its negative regulator, Mig6, alters signaling outcomes and results in variable drug sensitivity.</p

THE ROLE OF PROGESTERONE RECEPTOR MEMBRANE COMPONENT 1 IN RECEPTOR TRAFFICKING AND DISEASE

The progesterone receptor membrane component 1 (PGRMC1) is a multifunctional protein with a heme-binding domain that promotes cellular signaling via receptor trafficking, and is essential for some elements of tumor growth and metastasis. PGRMC1 is upregulated in breast, colon, lung and thyroid tumors. We expanded the analysis of PGRMC1 in the clinical setting, and report the first analysis of PGRMC1 in human oral cavity and ovarian tumors and found PGRMC1 to correlate with lung and ovarian cancer patient survival. Furthermore, we discovered a specific role for PGRMC1 in cancer stem cell viability. PGRMC1 directly associates with the epidermal growth factor (EGFR) in cancer cells, and we reviewed multiple signaling-associated pathways that are important in trafficking wild-type and mutant EGFR. To better understand the potential of PGRMC1 in receptor tyrosine kinase trafficking, we extended our research to the insulin receptor (IR). Changes in insulin signaling have been linked to multiple diseases, because IR plays a key role in glucose metabolism, cellular survival and proliferation. We found PGRMC1 to co-precipitate with IR in cancer cells and in an adipose model system. PGRMC1 increased IR plasma membrane levels in multiple cancer cell lines, and was also found to increase plasma membrane levels of two glucose transporters. Treatment with a PGRMC1 ligand significantly increased IR levels in human adipocytes. Moreover, we demonstrate that both insulin binding and glucose uptake are dependent on PGRMC1

Regulation of Egf Receptor Dynamics by Protein Tyrosine Phosphatases

The phosphorylated epidermal growth factor receptor (EGFR) initiates intracellular signaling processes that regulate cell growth, survival, and migration, and disregulated EGFR-mediated signaling occurs in many cancers. While the processes that lead to EGFR activation and phosphorylation have been studied in detail, quantitative aspects of the spatiotemporal regulation of EGFR by protein tyrosines phosphatases (PTPs) are not well understood. To begin to address this, we developed a new compartmentalized mechanistic model of EGFR phosphorylation dynamics and used it to interpret quantitative biochemical measurements to show that EGFR is dephosphorylated at the plasma membrane and in the cell interior with a time scale that is small compared to the time scales for EGFR internalization. By expanding our computational model and experimental data set, we went on to demonstrate that EGFR dephosphorylation at the plasma membrane surprisingly does not affect phosphorylation-dependent EGFR internalization because a separation of phospho-dependent time scales enables EGFR to enter clathrin-coated pits prior to being acted upon by PTPs. This same separation of time scales does, however, allow PTPs to control EGFR association with adapter proteins that regulate downstream signaling. Thus, our model provides new quantitative understanding of how EGFR participates in a number of simultaneous processes that compete for EGFR C-terminal phosphotyrosines. We went on to apply this new quantitative understanding of EGFR regulation by PTPs by developing predictive models to understand how such regulation might differentially impact the efficacy of antibodies and kinase inhibitors targeting EGFR. We also developed new computational models to quantitatively predict how receptor dephosphorylation kinetics as rapid as we found for EGFR might differentially control signaling initiated by receptor tyrosine kinases that dimerize in structurally distinct ways observed naturally among the family of receptor tyrosine kinases (RTKs). Ultimately, the new quantitative understanding of EGFR regulation by PTPs developed in this thesis significantly refines our understanding of the dynamics of EGFR-mediated signaling, provides a number of additional testable predictions related to fundamental aspects of EGFR signaling complex nucleation and efficacy of EGFR-targeted therapeutics, and offers a basic quantitative framework for exploring the regulation of other receptor tyrosine kinases by PTPs

Modulación de la actividad de EGFR en gioblastomas: nuevas aproximaciones terapéuticas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lecdtura: 23-01-2015Los glioblastomas (GBMs) son tumores cerebrales primarios muy agresivos y resistentes al tratamiento convencional con quimio y radioterapia. Dado que EGFR (del inglés Epidermal growth factor receptor) se encuentra alterado en el 50 % de los GBMs, representa actualmente una de las dianas terapéuticas más prometedoras en este tipo de tumores. Sin embargo, los inhibidores de la actividad quinasa de EGFR han generado escasos resultados en ensayos clínicos con pacientes de GBM, sin que exista una clara explicación para esta resistencia a dicha terapia. En este trabajo se ha evaluado la eficacia antitumoral de un inhibidor de EGFR de segunda generación denominado dacomitinib (PF299804, Pfizer), que se une de manera irreversible al receptor. Los resultados obtenidos confirman que dacomitinib es capaz de reducir la viabilidad celular, la autorenovación y la proliferación de las células de GBM con EGFR amplificado, con independencia de la expresión de la forma mutante EGFRvIII. Además, se ha observado una disminución notable de la tasa de crecimiento tumoral in vivo de dichas líneas de GBM tras la administración sistémica de dacomitinib a los ratones, coincidiendo con la reducción de los niveles de expresión de marcadores celulares característicos de célula madre. Sin embargo, nuestros resultados indican también que el efecto del inhibidor es reversible y que los tumores vuelven a crecer cuando se retira el tratamiento. Además, aunque en presencia de dacomitinib se produce una clara inhibición de la cascada de señalización inducida por EGFR, también se observa la acumulación del mismo, lo que podría provocar la activación de otras señales independientes de su actividad quinasa. Es por ello que el presente trabajo explora una estrategia novedosa y alternativa a dacomitinib. Nuestros resultados describen el papel fundamental de DYRK1A (del inglés Dual-specificity tyrosine-phosphorylation-regulated kinase 1A) en la regulación de la estabilidad de EGFR en las células de GBM. Así hemos comprobado que la inhibición de DYRK1A (ya sea farmacológicamente o genéticamente) es capaz de promover la degradación de EGFR en cultivos primarios de GBM, con una reducción notable de la capacidad de autorenovación de las células tumorales. Además, los resultados obtenidos sugieren que la supervivencia celular de un subgrupo de GBMs depende de la presencia de elevados niveles de EGFR en la superficie celular, ya que la inhibición de DYRK1A provoca un importante descenso de la carga tumoral. A la luz de los resultados obtenidos, se podría postular que la inhibición de EGFR con dacomitinib y el bloqueo de de DYRK1A representan una aproximación terapéutica prometedora en aquellos GBMs dependientes de EGFR, ya sea de manera individual o combinada, bloqueando toda la señalización activada por el receptor, tanto dependiente como independiente de su actividad quinasa.Glioblastomas (GBMs) are very aggressive primary brain tumors, which are resistant to conventional chemo and radiotherapy. Since EGFR (epidermal growth factor receptor) is altered in almost 50% of GBM, it currently represents one of the most promising therapeutic targets. However EGFR kinase activity inhibitors have produced poor results in clinical trials with GBM patients, with no clear explanation for the therapy resistance observed. Here it has been tested the antitumoral efficacy of a second-generation inhibitor: dacomitinib (PF299804, Pfizer) that binds in an irreversible way to the receptor. The results obtained confirm that dacomitinib is able to reduce the cell viability, the self-renewal and the proliferation of EGFR amplified GBM cells, independently of the EGFRvIII mutant form expression. Moreover we have observed a considerable decrease of the in vivo tumor growth rate of these EGFR amplified cell lines after systemic administration of dacomitinib to the mice, which provokes also a decrease in the expression of stem-cell-related markers. Nevertheless our results point out that the inhibitor effect is reversible and the tumors grow again when the treatment is interrupted. Moreover despite the clear inhibition of EGFR phosphorilation in the presence of the drug we observe also a clear accumulation of the receptor, which could provoke the activation of other signals that are independent of its kinase activity. For that reason we decided to analyze an alternative strategy to dacominib. Our results describe the basic role of DYRK1A (dual-specificity tyrosinephosphorylation- regulated kinase) in regulating the EGFR stability in GBM cells. We have observed that the inhibition of DYRK1A (pharmacologically or genetically) is able to promote the EGFR degradation in the GBM primary cell cultures, reducing the self-renewal capacity of tumorigenic cells. Moreover, the obtained results suggest that the cell survival of a subset of GBMs depends on the presence of high levels of EGFR on the cell surface, as the DYRK1A inhibition causes a profound decrease in tumor burden. In light of the obtained results it could be postulated that the inhibition of EGFR with dacomitinib and the blockade of DYRK1A (individually or combined) represent a promising therapeutic approach in those EGFR-dependent GBMs, blocking all the signals activated by the receptor, both dependent and independent of its kinase activity

Future Aspects of Tumor Suppressor Gene

Tumor suppressor genes (TSGs) and their signaling networks are fast growing areas in current biomedical science. These groups of genes, which are not limited to tumor suppression, play critical roles in many cellular activities. This book, "Future Aspects of Tumor Suppressor Genes", contains some fascinating fields, from basic to translational researches, in recent TSG studies. For example, several TSG signaling pathways are addressed in this book, and both mouse and Drosophila models used for the exploration of these genes are described based on the experimental evidence. A detailed review for current knowledge of microRNA studies in the regulation of tumor growth is introduced. Additionally, how natural compounds interfere with the progression of cancer development via TSG pathways is systemically summarized. Recent progresses in cell reprogramming and stemness transition processes regulated by TSG pathways are also included in this book

Future aspects of tumor suppressor gene

Tumor suppressor genes (TSGs) and their signaling networks are fast growing areas in current biomedical science. These groups of genes, which are not limited to tumor suppression, play critical roles in many cellular activities. This book, 'Future Aspects of Tumor Suppressor Genes', contains some fascinating fields, from basic to translational researches, in recent TSG studies. For example, several TSG signaling pathways are addressed in this book, and both mouse and Drosophila models used for the exploration of these genes are described based on the experimental evidence. A detailed review for current knowledge of microRNA studies in the regulation of tumor growth is introduced. Additionally, how natural compounds interfere with the progression of cancer development via TSG pathways is systemically summarized. Recent progresses in cell reprogramming and stemness transition processes regulated by TSG pathways are also included in this book.published_or_final_versio