Inter-receptor Crosstalk Research Articles

Alleviation of doxorubicin adverse effects via loading into various drug-delivery systems: a comparative study.

Aim: Drug resistance is still a significant barrier to effective hepatocellular carcinoma therapy. Address the issue of doxorubicin resistance and inter-receptor crosstalk various doxorubicin formulations were investigated. Methods: Hepatocellular carcinoma was carried out using 3-methylechloroanthrene. Animals were then treated with doxorubicin, liposomal doxorubicin, titanium-loaded doxorubicin (TiO2-Dox), lactoferrin-doxorubicin and PEGylated doxorubicin. Biochemical and molecular analyses were assessed. Results: Results have declared a significant alternation of both sodium and potassium concentrations upon 3-methylechloroanthrene administration. Arginase-I and α-L-Fucodinase tumor biomarkers were significantly elevated. C-myc, Hprt-1 and EGFR gene expression were over-expressed. Treatment with the aforementioned treatment regimens significantly modulated all measured parameters. Conclusion: TiO2-Dox, doxorubicin-lactoferrin and PEGylated doxorubicin could be a promising regimen in hepatocellular carcinoma and overcoming the problem ofdrug resistance.

ISY1-1 - Innate resistance in EGFR mutant non-small-cell lung cancer patients by co-activation of receptor tyrosine kinases(RTKs)

The MET ligand HGF in stromal cells provides an alternative signaling mechanism for EGFR by inducing inter-receptor cross talk with EphA2, CDCP1 or AXL. Src-homology 2 domain-containing phosphatase 2 (SHP2) also plays a role in RTK signaling and is required for MAPK activation. We studied the expression levels of stromal HGF and tumor RTKs: AXL, CDCP1, MET, and EphA2, as well as SHP2, and clinical outcome in baseline samples of 64 EGFR-mutant NSCLC patients (pts) treated with first-line EGFR TKI. Paraffin-embedded samples and the discrimination between tumor and stromal cells were performed by a pathologist. mRNA expression was analyzed by qRT-PCR. AXL and CDCP1 were among the genes that most significantly influenced treatment outcome. Median progression-free survival (PFS) was 14 months (mo) and 23.4 mo for pts with high and low AXL mRNA, respectively (p = 0.03), (HR: 2.05, p = 0.03). Median PFS was 9 mo and 20.2 mo for pts with high and low CDCP1 mRNA, respectively (p = 0.01), (HR: 2.1, p = 0.02). There were no significant differences in PFS according to levels of EPHA2 and MET in the tumor, and HGF in the stroma. Median PFS was 11.4 mo and 24 mo for pts with high and low SHP2 mRNA, respectively (p = 0.09) (HR: 2.4, p = 0.01). Median overall survival (OS) was 19.1 mo and 40.7 mo for pts with high and low AXL mRNA, respectively (p = 0.009) (HR: 2.9, p = 0.001). Median OS was 19.1 mo and 34.9 mo for pts with high and low CDCP1 mRNA, respectively (p = 0.03) (HR: 2.2, p = 0.03). In addition, differences in OS were noted according to levels of EPHA2, MET and SHP2. AXL and CDCP1 are adverse predictive markers of PFS and OS. It has been reported that overexpression of several RTKs can substitute EGFR signaling in EGFR mutant NSCLC pts. The findings show the need to scrutinize RTK levels in EGFR mutant NSCLC patients and, henceforth, the importance of tailored combinatory therapy with AXL inhibitors, or with Src inhibitors, in those pts with elevated CDCP1 (CDCP1 triggers SFK activation).

Unmet Medical Needs in Non-Small-Cell Lung Cancer Treatment: How to Design Pre-Emptive Combination Therapies

The rapidly expanding catalogue of human oncogenic mutations, coupled with difficulties in identifying the cellular targets of active compounds in phenotypic screens, has refocused drug discovery efforts on inhibitors of specific cellular proteins. This new ‘target-based’ approach has enjoyed some spectacular successes in several types of tumours, including non-small-cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) mutations occur in 17% of NSCLC patients, with notable response to single agent therapy. Unfortunately, all patients eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs), while complete remission rate to EGFR TKIs monotherapy is low. Priming BIM, a proapoptotic signalling BH3-only protein, induces sensitivity to erlotinib [Tarceva®] in EGFR-mutant cell lines. Synthetic lethal approaches and pre-emptive therapies based on the initial expression of BIM may significantly improve treatment outcomes. EGFR mutations result in transient pro-death imbalance of survival and apoptotic signalling in response to EGFR inhibition. Src homology 2 domain-containing phosphatase 2 is essential to the balance between extracellular signal-regulated kinase, phosphoinositide- 3-kinase/protein kinase B and signal transducer and activator of transcription 3 activity. Furthermore, stromal hepatocyte growth factor confers EGFR TKI resistance and induces inter-receptor crosstalk with Ephrin Type-A receptor 2, CDCP1, AXL, and JAK1. A better understanding of the complex cancer molecular biology of EGFR mutant lung cancer is crucial for development of effective treatment and design of successful future clinical studies.

148IN - Can we Do Better with Our Current Therapies for Nsclc? the Spanish Lung Cancer Group Approach

ABSTRACT Aim: In the SLCG large-scale screening, 16.6% of EGFR mutations were found, median PFS and OS for pts receiving erlotinib was 14 mos and 27 mos, respectively. The HR for the duration of PFS was 1.68 for the presence of L858R mutation in paired serum DNA (P = 0.02) (NEJM 2009; 361:958-67). Mutant EGFR NSCLC selectively activates STAT3 signaling which promotes cell survival and single EGFR inhibition can even enhance STAT3 activation. Growing evidence shows that targeting STAT3 or upstream or downstream components could improve the outcome. Hence, the resistance mechanisms should be revised, T790M is detected in 60% before therapy (reported by SLCG) and MET signaling can cause inter-receptor crosstalk with AXL, EPHA2, JAK1 and CDCP1. BIM mRNA expression is an independent predictive marker of response to erlotinib (reported by SLCG). High BIM can predict response and early adaptive resistance via IL6-STAT3-Bcl2. In those patients with low BIM, STAT3 can be activated through over-expression of AXL and EPHA2. Crosstalk between TGFb and NOTCH signaling can also be involved. Methods: Transcripts involving STAT3 signaling were examined from 80 EGFR mutant NSCLC pts, including BIM, STAT3 and SHP2. Also MET, AXL, Mer, EphA2 and Gas6 were analyzed. Together with TGFb and NOTCH constituents: ADAM17, NUMB, HES1, DUSP1, RBJ, LncRNA-ATB, SLUG, GLI and PKCi. Repurposing drugs, in combination with gefitinib, erlotinib and afatinib, were examined in cell cultures and in subcutaneous and orthotopic xenograft mice models. Results: Data will be presented on the correlation of BIM and SHP2 with regard to the mechanism of activation of STAT3 and the influence in response, PFS and OS in the 80 erlotinib-treated EGFR mutant NSCLC pts, and the influence of the above mentioned signaling constituents. Early data shows synergism of gefitinib with AXL inhibitor in the PC9-R cells. Also, activity of niclosamide and paclitaxel. Conclusions: Based on the results, the SLCG-GFCP will carry out a trial with erlotinib in combination with the most active repurposing drugs found, customized by BIM expression. Disclosure: All authors have declared no conflicts of interest.

Predicting resistance by selection of signaling pathways.

Epidermal growth factor receptor (EGFR) mutations occur in 17% of non-small-cell lung cancer (NSCLC) patients with notable response to single agent therapy but with low complete remission rate and, eventually, disease progression. Priming BIM, a pro-apoptotic signaling BH3-only protein, induces sensitivity to erlotinib in EGFR-mutant cell lines. Synthetic lethal approaches and preemptive therapies based on the initial expression of BIM may significantly improve the treatment outcome. EGFR mutations result in transient pro-death imbalance of survival and apoptotic signaling in response to EGFR inhibition. SHP2 is essential to the balance between ERK and the phosphoinositide-3-kinase (PI3K)/AKT and signal transducer activator of transcription (STAT) activity, while mTOR can be an additional marker for patients with high BIM expression. Furthermore, stromal hepatocyte growth factor (HGF) confers EGFR tyrosine kinase inhibitor (TKI) resistance and induces interreceptor crosstalk with integrin-b4, Eph2, CUB domain-containing protein-1 (CDCP1), AXL and JAK1. Only by understanding better, and in more depth, complex cancer molecular biology will we have the information that will help us to design strategies to augment efficacy of EGFR TKIs and offer our patients the best, most correct therapeutic option.

Astrocytes Modulate a Postsynaptic NMDA–GABAA-Receptor Crosstalk in Hypothalamic Neurosecretory Neurons

A dynamic balance between the excitatory and inhibitory neurotransmitters glutamate and GABA is critical for maintaining proper neuronal activity in the brain. This balance is partly achieved via presynaptic interactions between glutamatergic and GABA(A)ergic synapses converging into the same targets. Here, we show that in hypothalamic magnocellular neurosecretory neurons (MNCs), a direct crosstalk between postsynaptic NMDA receptors (NMDARs) and GABA(A) receptors (GABA(A)Rs) contributes to the excitatory/inhibitory balance in this system. We found that activation of NMDARs by endogenous glutamate levels controlled by astrocyte glutamate transporters, evokes a transient and reversible potentiation of postsynaptic GABA(A)Rs. This inter-receptor crosstalk is calcium-dependent and involves a kinase-dependent phosphorylation mechanism, but does not require nitric oxide as an intermediary signal. Finally, we found the NMDAR-GABA(A)R crosstalk to be blunted in rats with heart failure, a pathological condition in which the hypothalamic glutamate-GABA balance is tipped toward an excitatory predominance. Together, our findings support a novel form of glutamate-GABA interactions in MNCs, which involves crosstalk between NMDA and GABA(A) postsynaptic receptors, whose strength is controlled by the activity of local astrocytes. We propose this inter-receptor crosstalk to act as a compensatory, counterbalancing mechanism to dampen glutamate-mediated overexcitation. Finally, we propose that an uncoupling between NMDARs and GABA(A)Rs may contribute to exacerbated neuronal activity and, consequently, sympathohumoral activation in such disease conditions as heart failure.

Epidermal Growth Factor Receptor (EGFR) Crosstalks in Liver Cancer

Hepatocarcinogenesis is a complex multistep process in which many different molecular pathways have been implicated. Hepatocellular carcinoma (HCC) is refractory to conventional chemotherapeutic agents, and the new targeted therapies are meeting with limited success. Interreceptor crosstalk and the positive feedback between different signaling systems are emerging as mechanisms of targeted therapy resistance. The identification of such interactions is therefore of particular relevance to improve therapeutic efficacy. Among the different signaling pathways activated in hepatocarcinogenesis the epidermal growth factor receptor (EGFR) system plays a prominent role, being recognized as a “signaling hub” where different extracellular growth and survival signals converge. EGFR can be transactivated in response to multiple heterologous ligands through the physical interaction with multiple receptors, the activity of intracellular kinases or the shedding of EGFR-ligands. In this article we review the crosstalk between the EGFR and other signaling pathways that could be relevant to liver cancer development and treatment.

Signaling through the Na/K-ATPase: implications for cardiac fibrosis

the cardiotonic effects of digitalis have been exploited in clinical practice for many years. However, while the existence of endogenous digitalis-like substances (also called cardiotonic steroids) has been accepted for some time, their exact chemical nature has been only recently demonstrated ([1][

5‐HT induces threonine (T375) phosphorylation of ADAM17/TACE cytoplasmic tail

We have shown recently that TACE transactivates epidermal growth factor receptor (EGFR), and induces ERK phosphorylation (ERK‐P) and cellular proliferation upon 5‐HT2A receptor stimulation in renal mesangial cells.In this study our aim was to identify kinases responsible for TACE phosphorylation and potential activation during inter‐receptor cross‐talk. We detected robust threonine phosphorylation of TACE as early as 6 minutes following 5‐HT stimulation using a phospho‐threonine/proline antibody. This suggested phosphorylation of threonine 375, as this is the only threonine next to a proline present in the cytoplasmic tail of TACE. Using chemical inhibitors (naltrindole and PDK1/Akt/Flt dual pathway inhibitor) we showed that ERK phosphorylation is PDK1 dependent. Additionally, a MAPK cascade seems to have a role in TACE phosphorylation in that a MEK inhibitor (10 μM of PD989059) significantly attenuated 1 μM 5‐HT‐induced TACE phosphorylation. To study kinase‐induced activation of the enzyme we used a quenched fluorogenic TACE substrate. We found attenuation of 5‐HT‐induced TACE activity in PD989059‐pretreated cells. No serine phosphorylation of TACE upon 5‐HT stimulation was detected. Our data suggest that (1) either MEK itself or members of the MAPK family can be (directly or indirectly) responsible for TACE phosphorylation and activation during inter‐receptor crosstalk, and (2) multiple kinases contribute to TACE phosphorylation.This work was supported by NIH DK070054‐03 to M.G., T32 DK07752‐08 to C.L.R., NIH NCCR P20 RR016434 to P.G., NIH R01 DK55524 to L.M.L. and REAP from the VA Research Service.

Membrane‐anchored growth factors, the epidermal growth factor family: Beyond receptor ligands

The epidermal growth factor (EGF) family and the EGF receptor (EGFR, ErbB) tyrosine kinase family have been spearheading the studies of signal transduction events that determine cell fate and behavior in vitro and in vivo. The EGFR family and their signaling pathways are giving us tremendous advantages in developing fascinating molecular target strategies for cancer therapy. Currently, two important types of EGFR inhibitors are in clinical use: neutralizing antibodies of EGFR or ErbB2, and synthetic small compounds of tyrosine kinase inhibitors designed for receptors. On the other hand, basic research of the EGF family ligands presents new challenges as membrane-anchored growth factors. All members of the EGF family have important roles in development and diseases and are shed from the plasma membrane by metalloproteases. The ectodomain shedding of the ligands has emerged as a critical component in the functional transactivation of EGFRs in interreceptor cross-talk in response to various shedding stimulants such as G-protein coupled receptor agonists, growth factors, cytokines, and various physicochemical stresses. Among the EGFR-ligands, heparin-binding EGF-like growth factor (HB-EGF) is a prominent ligand in our understanding of the pathophysiological roles of ectodomain shedding in cancer, wound healing, cardiac diseases, etc. Here we focus on ectodomain shedding of the EGF family ligands, especially HB-EGF by disintegrin and metalloproteases, which are not only key events of receptor cross talk, but also novel intercellular signaling by their carboxy-terminal fragments to regulate gene expression directly.

5-HT2A Receptor Induces ERK Phosphorylation and Proliferation through ADAM-17 Tumor Necrosis Factor-α-converting Enzyme (TACE) Activation and Heparin-bound Epidermal Growth Factor-like Growth Factor (HB-EGF) Shedding in Mesangial Cells

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.

Epithelial Cell Signaling in Helicobacter pylori Infection

The human microbial pathogen Helicobacter pylori colonises the stomach of more than half of the worlds population. The microorganism can induce chronic gastritis, peptic ulceration and more rarely, gastric adenocarcinoma. Highly virulent H. pylori strains carry a cag pathogenicity island (cag PAI), which encodes proteins involved in a specialised type IV secretion system (T4SS). H. pylori induces T4SS-dependent and -independent processes by which H. pylori takes direct command of gastric epithelial cell signaling. The H. pylori effector protein cytotoxin associated gene A (CagA), which is translocated via the T4SS into epithelial cells, contributes to the modulation of cellular functions. In addition, H. pylori transactivates the EGFR, a process involving inter-receptor cross talk and extracellular ADAM metalloproteinase cleavage of membrane bound EGFR ligands. The multiple signal transduction pathways activated during H. pylori infection lead to a complex series of events promoting inappropriate inflammatory responses, epithelial hyperproliferation, epithelial survival and transformation. The H. pylori induced epithelial cellular changes, as well as chemopreventative therapeutic strategies, will be introduced in this review. Keywords: Helicobacter pylori, CagA, epithelial proliferation, NF-kB, EGFR, ADAM metalloproteinases, c-Met

Src-mediated Inter-receptor Cross-talk between the Na+/K+-ATPase and the Epidermal Growth Factor Receptor Relays the Signal from Ouabain to Mitogen-activated Protein Kinases

Binding of ouabain to Na(+)/K(+)-ATPase activates tyrosine phosphorylation of the epidermal growth factor receptor (EGFR), Src, and p42/44 mitogen-activated protein kinases (MAPKs) in both cardiac myocytes and A7r5 cells. Here, we explored the roles of Src and the EGFR in the ouabain-invoked pathways that lead to the activation of MAPKs. Exposure of A7r5 and LLC-PK1 cells to ouabain caused a dose-dependent inhibition of Na(+)/K(+)-ATPase activity, which correlated well with ouabain-induced activation of Src and MAPKs in these cells. Immunoprecipitation experiments showed that ouabain stimulated Src binding to Na(+)/K(+)-ATPase in a dose- and time-dependent manner and increased phosphorylation of Src at Tyr(418) but had no effect on Tyr(529) phosphorylation. Ouabain failed to activate MAPKs in A7r5 cells that were pretreated with the Src inhibitor PP2 and in SYF cells in which Src family kinases are knocked out. Preincubation with AG1478, but not AG1295, also blocked the effects of ouabain on p42/44 MAPKs in A7r5 cells. Significantly, both herbimycin A and PP2 abrogated ouabain-induced but not epidermal growth factor-induced Src binding to the EGFR and the subsequent EGFR tyrosine phosphorylation. Ouabain also failed to affect tyrosine phosphorylation of the EGFR in SYF cells. In addition, unlike epidermal growth factor, ouabain did not increase EGFR autophosphorylation at Tyr(1173). These findings clearly indicate that ouabain transactivates the EGFR by activation of Src and stimulation of Src binding to the EGFR. Furthermore, we found that the transactivated EGFR was capable of recruiting and phosphorylating the adaptor protein Shc. This resulted in increased binding of another adaptor protein Grb2 to the Src-EGFR complex and the subsequent activation of Ras and MAPKs. Taken together, these new findings suggest that Src mediates the inter-receptor cross-talk between Na(+)/K(+)-ATPase and the EGFR to transduce the signals from ouabain to the Ras/MAPK cascade.

The Cellular Response to Neuregulins Is Governed by Complex Interactions of the erbB Receptor Family

Deregulated signaling by the four members of the epidermal growth factor receptor tyrosine kinase family (erbB family) is implicated in the genesis or progression of human cancers. However, efforts to analyze signaling by these receptors have been hampered by the diversity of ligands and extensive interreceptor cross talk. We have expressed the four human erbB family receptors, singly and in pairwise combinations, in a pro-B-lymphocyte cell line (Ba/F3) and investigated the range of interactions activated by the epidermal growth factor homology domain of the agonist neuregulin beta. The results provide the first comprehensive analysis of the response of this receptor family to a single peptide agonist. This peptide induced complex patterns of receptor tyrosine phosphorylation and regulation of Ba/F3 cell survival and proliferation. These data demonstrate the existence of several previously undocumented receptor interactions driven by neuregulin.

Differential regulation of accessory mitogenic signaling receptors by the T cell antigen receptor

In addition to the antigen receptor, resting T cells express a number of receptors that can be stimulated to generate proliferative signals. These “accessory” receptors require co-expression of the T cell receptor (TCR), suggesting that they channel their signals via secondary activation of the signal transduction function of the CD3-TCR complex. Little is known about how different receptors control each other's function when one or more stimuli are presented at the same time. In order to study the regulation of accessory receptors by the CD3-TCR and vice versa, we have investigated the activation of the CD2 cell adhesion molecule receptor and the pertussis toxin receptor, a 43 kDa plasma membrane protein. Both receptors can activate signal transduction pathways in T cells similar to that of the CD3-TCR, including increases in Ca 2+ and phosphatidylinositol turnover. They are also similar in that they utilize the antigen receptor to transmit their signals to the cell since CD3-TCR(−) mutants cannot be activated via either CD2 or the toxin receptor. We have previously shown that submaximal stimulation of the CD3-TCR blocks second messenger generation and proliferation in response to pertussis toxin. This heterologous desensitization was unidirectional since activation of the toxin receptor had no effect on CD3-TCR function. Here we extend these studies to show that activation of both CD2 and the toxin receptor led to rapid tyrosine phosphorylation of three similar proteins. Submaximal stimulation of the CD3-TCR completely inhibited toxin receptor-stimulated tyrosine protein kinase activity but did not desensitize CD2 function as determined by activation of tyrosine protein phosphorylation. Furthermore, CD2 stimulation did not lead to desensitization of the pertussis toxin receptor. These data support a system of complex regulatory relationships between different signaling receptors and suggest a model for signal integration and inter-receptor cross-talk in T cell activation.