Concomitant Tumor Expression of EGFR and TATI/SPINK1 Associates with Better Prognosis in Colorectal Cancer

Background

Epidermal growth factor receptor (EGFR) activation plays a role in colorectal cancer (CRC) carcinogenesis, and anti-EGFR drugs are used in treatment of advanced CRC. One of the EGFR ligands is tumor-associated trypsinogen inhibitor TATI, also called serine protease inhibitor Kazal type1 (SPINK 1), which we recently showed to be an independent prognostic marker in CRC.

Methods

We studied the prognostic value of immunohistochemical expression of EGFR and concomitant expression of EGFR and TATI/SPINK1 in a series of 619 colorectal cancer patients.

Results

Of the samples, 92% were positive for EGFR. EGFR+/TATI+ was seen in 62.8%, EGFR+/TATI− in 29.5%, EGFR−/TATI+ in 4.9%, and EGFR−/TATI− in 2.7% of patients. EGFR expression correlated with WHO grade (p = 0.040). In univariate analysis, EGFR expression correlated with favourable survival (p = 0.006). EGFR+/TATI+ patients showed better survival than did those with other combinations (p<0.001). In multivariate analysis, EGFR+/TATI+ was an independent prognostic factor of favourable prognosis (p<0.001).

Conclusion

Concomitant positivity of EGFR and TATI/SPINK1 predicts favourable prognosis in CRC.     

Selection of DNA Aptamers against Glioblastoma Cells with High Affinity and Specificity

Background

Glioblastoma is the most common and most lethal form of brain tumor in human. Unfortunately, there is still no effective therapy to this fatal disease and the median survival is generally less than one year from the time of diagnosis. Discovery of ligands that can bind specifically to this type of tumor cells will be of great significance to develop early molecular imaging, targeted delivery and guided surgery methods to battle this type of brain tumor.

Methodology/Principal Findings

We discovered two target-specific aptamers named GBM128 and GBM131 against cultured human glioblastoma cell line U118-MG after 30 rounds selection by a method called cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX). These two aptamers have high affinity and specificity against target glioblastoma cells. They neither recognize normal astraglial cells, nor do they recognize other normal and cancer cell lines tested. Clinical tissues were also tested and the results showed that these two aptamers can bind to different clinical glioma tissues but not normal brain tissues. More importantly, binding affinity and selectivity of these two aptamers were retained in complicated biological environment.

Conclusion/Significance

The selected aptamers could be used to identify specific glioblastoma biomarkers. Methods of molecular imaging, targeted drug delivery, ligand guided surgery can be further developed based on these ligands for early detection, targeted therapy, and guided surgery of glioblastoma leading to effective treatment of glioblastoma.     

HER2 oncogenic function escapes EGFR tyrosine kinase inhibitors via activation of alternative HER receptors in breast cancer cells

Background

The response rate to EGFR tyrosine kinase inhibitors (TKIs) may be poor and unpredictable in cancer patients with EGFR expression itself being an inadequate response indicator. There is limited understanding of the mechanisms underlying this resistance. Furthermore, although TKIs suppress the growth of HER2-overexpressing breast tumor cells, they do not fully inhibit HER2 oncogenic function at physiological doses.

Methodology and Principal Findings

Here we have provided a molecular mechanism of how HER2 oncogenic function escapes TKIs'' inhibition via alternative HER receptor activation as a result of autocrine ligand release. Using both Förster Resonance Energy Transfer (FRET) which monitors in situ HER receptor phosphorylation as well as classical biochemical analysis, we have shown that the specific tyrosine kinase inhibitors (TKIs) of EGFR, AG1478 and Iressa (Gefitinib) decreased EGFR and HER3 phosphorylation through the inhibition of EGFR/HER3 dimerization. Consequent to this, we demonstrate that cleavage of HER4 and dimerization of HER4/HER2 occur together with reactivation of HER3 via HER2/HER3, leading to persistent HER2 phosphorylation in the now resistant, surviving cells. These drug treatment–induced processes were found to be mediated by the release of ligands including heregulin and betacellulin that activate HER3 and HER4 via HER2. Whereas an anti-betacellulin antibody in combination with Iressa increased the anti-proliferative effect in resistant cells, ligands such as heregulin and betacellulin rendered sensitive SKBR3 cells resistant to Iressa.

Conclusions and Significance

These results demonstrate the role of drug-induced autocrine events leading to the activation of alternative HER receptors in maintaining HER2 phosphorylation and in mediating resistance to EGFR tyrosine kinase inhibitors (TKIs) in breast cancer cells, and hence specify treatment opportunities to overcome resistance in patients.     

Expression analysis of the ligands for the Natural Killer cell receptors NKp30 and NKp44

Background

The natural cytotoxicity receptors (NCR) are important to stimulate the activity of Natural Killer (NK) cells against transformed cells. Identification of NCR ligands and their level of expression on normal and neoplastic cells has important implications for the rational design of immunotherapy strategies for cancer.

Methodology/Principal Findings

Here we analyze the expression of NKp30 ligand and NKp44 ligand on 30 transformed or non-transformed cell lines of different origin. We find intracellular and surface expression of these two ligands on almost all cell lines tested. Expression of NKp30 and NKp44 ligands was variable and did not correlate with the origin of the cell line. Expression of NKp30 and NKp44 ligand correlated with NKp30 and NKp44-mediated NK cell lysis of tumor cells, respectively. The surface expression of NKp30 ligand and NKp44 ligand was sensitive to trypsin treatment and was reduced in cells arrested in G₂/M phase.

Conclusion/Significance

These data demonstrate the ubiquitous expression of the ligands for NKp30 and NKp44 and give an important insight into the regulation of these ligands.     

Clustered gene expression changes flank targeted gene loci in knockout mice

Background

Gene expression profiling using microarrays is a powerful technology widely used to study regulatory networks. Profiling of mRNA levels in mutant organisms has the potential to identify genes regulated by the mutated protein.

Methodology/Principle Findings

Using tissues from multiple lines of knockout mice we have examined genome-wide changes in gene expression. We report that a significant proportion of changed genes were found near the targeted gene.

Conclusions/Significance

The apparent clustering of these genes was explained by the presence of flanking DNA from the parental ES cell. We provide recommendations for the analysis and reporting of microarray data from knockout mice     

Solution Structure of Tensin2 SH2 Domain and Its Phosphotyrosine-Independent Interaction with DLC-1

Background

Src homology 2 (SH2) domain is a conserved module involved in various biological processes. Tensin family member was reported to be involved in tumor suppression by interacting with DLC-1 (deleted-in-liver-cancer-1) via its SH2 domain. We explore here the important questions that what the structure of tensin2 SH2 domain is, and how it binds to DLC-1, which might reveal a novel binding mode.

Principal Findings

Tensin2 SH2 domain adopts a conserved SH2 fold that mainly consists of five β-strands flanked by two α-helices. Most SH2 domains recognize phosphorylated ligands specifically. However, tensin2 SH2 domain was identified to interact with nonphosphorylated ligand (DLC-1) as well as phosphorylated ligand.

Conclusions

We determined the solution structure of tensin2 SH2 domain using NMR spectroscopy, and revealed the interactions between tensin2 SH2 domain and its ligands in a phosphotyrosine-independent manner.     

EGFR protein overexpression correlates with chromosome 7 polysomy and poor prognostic parameters in clear cell renal cell carcinoma

Background

The role of epidermal growth factor (EGF) and its receptor (EGFR) in the pathogenesis and progression of various malignant tumors has long been known, but there is still disagreement concerning prognostic significance of EGFR expression in clear cell renal cell carcinoma (CCRCC). The present study was designed to analyze more objectively the protein EGFR expression in CCRCC and to compare its value with EGFR gene copy number changes and clinicopathologic characteristics including patient survival.

Methods

The protein EGFR expression was analyzed immunohistochemically on 94 CCRCC, and gene copy number alterations of EGFR by FISH analysis on 41 CCRCC selected according to distinct membrane EGFR staining.

Results

Membrane EGFR expression in tumor cells was heterogeneous with respect to the proportion of positive cells and staining intensity. FISH analysis did not reveal EGFR gene amplification, while polysomy of chromosome 7 found in 41% was associated with higher EGFR membrane expression. Moreover, EGFR overexpression was associated with a higher nuclear grade, larger tumor size and shorter patient''s survival, while there was no connection with pathological stage.

Conclusion

In conclusion, the protein expression of EGFR had an impact on prognosis in patients with CCRCC, while an increased copy number of chromosome 7 could be the possible reason for EGFR protein overexpression in the absence of gene amplification.     

Planning and monitoring of patients for electrical cardioversion for atrial fibrillation

Objectives

This study evaluated the waiting list for elective electrical cardioversion (ECV) for persistent atrial fibrillation (AF), focusing on when and why procedures were postponed. We compared the effects of management of the waiting list conducted by physicians versus management by nurse practitioners (NPs) and we evaluated the safety of our anticoagulating policy by means of bleeding or thromboembolic complications during and after ECV.

Background

Not all patients selected for ECV receive their treatment at the first planned instance due to a variety of reasons. These reasons are still undocumented.

Methods

We evaluated 250 consecutive patients with persistent AF admitted to our clinic for elective ECV.

Results

Within 5 to 6 weeks, 186 of 242 patients (77%) received ECV. The main reason for postponing an ECV was an inadequate international normalised ratio (INR); other reasons included spontaneous sinus rhythm and switch to rate control. A total of 23 of the 147 patients (16%) managed by the research physician were postponed due to an inadequate INR at admission versus 4 out of 98 patients (4%) managed by NPs (p = 0.005)

Conclusion

An inadequate INR is the main reason for postponing an ECV. Management of ECV by NPs is safe and leads to less postponing on admission.     

Neuroprotective effect of transplanted human embryonic stem cell-derived neural precursors in an animal model of multiple sclerosis

Background

Multiple sclerosis (MS) is an immune mediated demyelinating disease of the central nervous system (CNS). A potential new therapeutic approach for MS is cell transplantation which may promote remyelination and suppress the inflammatory process.

Methods

We transplanted human embryonic stem cells (hESC)-derived early multipotent neural precursors (NPs) into the brain ventricles of mice induced with experimental autoimmune encephalomyelitis (EAE), the animal model of MS. We studied the effect of the transplanted NPs on the functional and pathological manifestations of the disease.

Results

Transplanted hESC-derived NPs significantly reduced the clinical signs of EAE. Histological examination showed migration of the transplanted NPs to the host white matter, however, differentiation to mature oligodendrocytes and remyelination were negligible. Time course analysis of the evolution and progression of CNS inflammation and tissue injury showed an attenuation of the inflammatory process in transplanted animals, which was correlated with the reduction of both axonal damage and demyelination. Co-culture experiments showed that hESC-derived NPs inhibited the activation and proliferation of lymph node–derived T cells in response to nonspecific polyclonal stimuli.

Conclusions

The therapeutic effect of transplantation was not related to graft or host remyelination but was mediated by an immunosuppressive neuroprotective mechanism. The attenuation of EAE by hESC-derived NPs, demonstrated here, may serve as the first step towards further developments of hESC for cell therapy in MS.     

Determination of EGFR Endocytosis Kinetic by Auto-Regulatory Association of PLD1 with μ2

Background

Upon ligand binding, cell surface signaling receptors are internalized through a process tightly regulated by endocytic proteins and adaptor protein 2 (AP2) to orchestrate them. Although the molecular identities and roles of endocytic proteins are becoming clearer, it is still unclear what determines the receptor endocytosis kinetics which is mainly regulated by the accumulation of endocytic apparatus to the activated receptors.

Methodology/Principal Findings

Here we employed the kinetic analysis of endocytosis and adaptor recruitment to show that μ2, a subunit of AP2 interacts directly with phospholipase D (PLD)1, a receptor-associated signaling protein and this facilitates the membrane recruitment of AP2 and the endocytosis of epidermal growth factor receptor (EGFR). We also demonstrate that the PLD1-μ2 interaction requires the binding of PLD1 with phosphatidic acid, its own product.

Conclusions/Significance

These results suggest that the temporal regulation of EGFR endocytosis is achieved by auto-regulatory PLD1 which senses the receptor activation and triggers the translocation of AP2 near to the activated receptor.     

Cell Surface Protein Disulfide Isomerase Regulates Natriuretic Peptide Generation of Cyclic Guanosine Monophosphate

Rationale

The family of natriuretic peptides (NPs), including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), exert important and diverse actions for cardiovascular and renal homeostasis. The autocrine and paracrine functions of the NPs are primarily mediated through the cellular membrane bound guanylyl cyclase-linked receptors GC-A (NPR-A) and GC-B (NPR-B). As the ligands and receptors each contain disulfide bonds, a regulatory role for the cell surface protein disulfide isomerase (PDI) was investigated.

Objective

We utilized complementary in vitro and in vivo models to determine the potential role of PDI in regulating the ability of the NPs to generate its second messenger, cyclic guanosine monophosphate.

Methods and Results

Inhibition of PDI attenuated the ability of ANP, BNP and CNP to generate cGMP in human mesangial cells (HMCs), human umbilical vein endothelial cells (HUVECs), and human aortic smooth muscle cells (HASMCs), each of which were shown to express PDI. In LLC-PK1 cells, where PDI expression was undetectable by immunoblotting, PDI inhibition had a minimal effect on cGMP generation. Addition of PDI to cultured LLC-PK1 cells increased intracellular cGMP generation mediated by ANP. Inhibition of PDI in vivo attenuated NP-mediated generation of cGMP by ANP. Surface Plasmon Resonance demonstrated modest and differential binding of the natriuretic peptides with immobilized PDI in a cell free system. However, PDI was shown to co-localize on the surface of cells with GC-A and GC-B by co-immunoprecpitation and immunohistochemistry.

Conclusion

These data demonstrate for the first time that cell surface PDI expression and function regulate the capacity of natriuretic peptides to generate cGMP through interaction with their receptors.     

Cortactin Is a Substrate of Activated Cdc42-Associated Kinase 1 (ACK1) during Ligand-induced Epidermal Growth Factor Receptor Downregulation

Background

Epidermal growth factor receptor (EGFR) internalization following ligand binding controls EGFR downstream pathway signaling activity. Internalized EGFR is poly-ubiquitinated by Cbl to promote lysosome-mediated degradation and signal downregulation. ACK1 is a non-receptor tyrosine kinase that interacts with ubiquitinated EGFR to facilitate EGFR degradation. Dynamic reorganization of the cortical actin cytoskeleton controlled by the actin related protein (Arp)2/3 complex is important in regulating EGFR endocytosis and vesicle trafficking. How ACK1-mediated EGFR internalization cooperates with Arp2/3-based actin dynamics during EGFR downregulation is unclear.

Methodology/Principal Findings

Here we show that ACK1 directly binds and phosphorylates the Arp2/3 regulatory protein cortactin, potentially providing a direct link to Arp2/3-based actin dynamics during EGFR degradation. Co-immunoprecipitation analysis indicates that the cortactin SH3 domain is responsible for binding to ACK1. In vitro kinase assays demonstrate that ACK1 phosphorylates cortactin on key tyrosine residues that create docking sites for adaptor proteins responsible for enhancing Arp2/3 nucleation. Analysis with phosphorylation-specific antibodies determined that EGFR-induced cortactin tyrosine phosphorylation is diminished coincident with EGFR degradation, whereas ERK1/2 cortactin phosphorylation utilized in promoting activation of the Arp2/3 regulator N-WASp is sustained during EGFR downregulation. Cortactin and ACK1 localize to internalized vesicles containing EGF bound to EGFR visualized by confocal microscopy. RNA interference and rescue studies indicate that ACK1 and the cortactin SH3 domain are essential for ligand-mediated EGFR internalization.

Conclusions/Significance

Cortactin is a direct binding partner and novel substrate of ACK1. Tyrosine phosphorylation of cortactin by ACK1 creates an additional means to amplify Arp2/3 dynamics through N-WASp activation, potentially contributing to the overall necessary tensile and/or propulsive forces utilized during EGFR endocytic internalization and trafficking involved in receptor degradation.     

Systematic Characterisation of Cellular Localisation and Expression Profiles of Proteins Containing MHC Ligands

Background

Presentation of peptides on Major Histocompatibility Complex (MHC) molecules is the cornerstone in immune system activation and increased knowledge of the characteristics of MHC ligands and their source proteins is highly desirable.

Methodology/Principal Finding

In the present large-scale study, we used a large data set of proteins containing experimentally identified MHC class I or II ligands and examined the proteins according to their expression profiles at the mRNA level and their Gene Ontology (GO) classification within the cellular component ontology. Proteins encoded by highly abundant mRNA were found to be much more likely to be the source of MHC ligands. Of the 2.5% most abundant mRNAs as much as 41% of the proteins encoded by these mRNAs contained MHC class I ligands. For proteins containing MHC class II ligands, the corresponding percentage was 11%. Furthermore, we found that most proteins containing MHC class I ligands were localised to the intracellular parts of the cell including the cytoplasm and nucleus. MHC class II ligand donors were, on the other hand, mostly membrane proteins.

Conclusions/Significance

The results contribute to the ongoing debate concerning the nature of MHC ligand-containing proteins and can be used to extend the existing methods for MHC ligand predictions by including the source protein''s localisation and expression profile. Improving the current methods is important in the growing quest for epitopes that can be used for vaccine or diagnostic purposes, especially when it comes to large DNA viruses and cancer.     

Comparative analysis of the heptahelical transmembrane bundles of G protein-coupled receptors

Background

G protein-coupled receptors represent a large family of eukaryotic membrane proteins, and are involved in almost all physiological processes in humans. Recent advances in the crystallographic study of these receptors enable a detailed comparative analysis of the commonly shared heptahelical transmembrane bundle. Systematic comparison of the bundles from a variety of receptors is indispensable for understanding not only of the structural diversification optimized for the binding of respective ligands but also of the structural conservation required for the common mechanism of activation accompanying the interaction changes among the seven helices.

Methodology/Principal Findings

We have examined the bundles of 94 polypeptide chains from almost all available structures of 11 receptors, which we classified into either inactivated chain or activated chain, based on the type of bound ligand. For the inactivated chains, superposition of 200 residue bundles by secondary structure matching demonstrated that the bound ligands share a laterally limited cavity in the extracellular section of the bundle. Furthermore, a distinct feature was found for helix III of bovine rhodopsin, which might have evolved to lower its activity in the presence of 11-cis-retinal, to a level that other receptors could hardly achieve with any currently available ligands.

Conclusions/Significance

Systematic analysis described here would be valuable for understanding of the rearrangement of seven helices which depends on the ligand specificity and activation state of the receptors.     

Identification of fluorescent compounds with non-specific binding property via high throughput live cell microscopy

Introduction

Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved.

Method

Three mammalian cell lines with diverse genetic backgrounds were used to screen a combinatorial fluorescence library via high throughput live cell microscopy for potential ligands with high in- and out-flux properties. The binding properties of ligands identified from the first screen were subsequently validated on plant root hair. A correlative analysis was then performed between each ligand and its corresponding physiochemical and structural properties.

Results

The non-stickiness property of each ligand was quantified as a function of the temporal uptake and retention on a cell-by-cell basis. Our data shows that (i) mammalian systems can serve as a pre-screening tool for complex plant species that are not amenable to high-throughput imaging; (ii) retention and spatial localization of chemical compounds vary within and between each cell line; and (iii) the structural similarities of compounds can infer their non-specific binding properties.

Conclusion

We have validated a protocol for identifying chemical compounds with non-specific binding properties that is testable across diverse species. Further analysis reveals an overlap between the non-stickiness property and the structural similarity of compounds. The net result is a more robust screening assay for identifying desirable ligands that can be used to monitor intracellular localization. Several new applications of the screening protocol and results are also presented.     

Common and Rare EGFR and KRAS Mutations in a Dutch Non-Small-Cell Lung Cancer Population and Their Clinical Outcome

Introduction

In randomly assigned studies with EGFR TKI only a minor proportion of patients with NSCLC have genetically profiled biopsies. Guidelines provide evidence to perform EGFR and KRAS mutation analysis in non-squamous NSCLC. We explored tumor biopsy quality offered for mutation testing, different mutations distribution, and outcome with EGFR TKI.

Patient and Methods

Clinical data from 8 regional hospitals were studied for patient and tumor characteristics, treatment and overall survival. Biopsies sent to the central laboratory were evaluated for DNA quality and subsequently analyzed for mutations in exons 18–21 of EGFR and exon 2 of KRAS by bidirectional sequence analysis.

Results

Tumors from 442 subsequent patients were analyzed. For 74 patients (17%) tumors were unsuitable for mutation analysis. Thirty-eight patients (10.9%) had EGFR mutations with 79% known activating mutations. One hundred eight patients (30%) had functional KRAS mutations. The mutation spectrum was comparable to the Cosmic database. Following treatment in the first or second line with EGFR TKI median overall survival for patients with EGFR (n = 14), KRAS (n = 14) mutations and wild type EGFR/KRAS (n = 31) was not reached, 20 and 9 months, respectively.

Conclusion

One out of every 6 tumor samples was inadequate for mutation analysis. Patients with EGFR activating mutations treated with EGFR-TKI have the longest survival.     

CXCL12/CXCR4 Axis Triggers the Activation of EGF Receptor and ERK Signaling Pathway in CsA-Induced Proliferation of Human Trophoblast Cells

Introduction

Our previous study has demonstrated Cyclosporin A (CsA) promotes the proliferation of human trophoblast cells. Therefore, we further investigate the intracellular signaling pathway involved in the CsA-induced proliferation of human trophoblast cells.

Methods

Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the regulation of CsA on CXCL12 secretion in human trophoblast cells. Immunofluorescence analysis and western blotting analysis were used to investigate the role of CXCL12/CXCR4 axis in the CsA-induced epidermal growth factor receptor (EGFR) phosphorylation in human trophoblast cells. 5-bromo-2′-deoxyuridine (BrdU) cell proliferation assay was performed to analyze the involvement of EGFR and its downstream extracellular signal-regulated protein kinase (ERK) signaling pathway in the CsA-induced proliferation of human trophoblast cells.

Results

Low concentration of CsA promoted the secretion of CXCL12, and recombinant human CXCL12 promoted the phosphorylation of EGFR in primary human trophoblast cells and choriocarcinoma cell line JEG-3. The inhibition of CXCL12 or CXCR4 by either neutralizing antibodies or small interfering RNA (siRNA) could completely block the CsA-induced EGFR phosphorylation. The CsA-induced proliferation of human trophoblast cells was effectively abrogated by the EGFR inhibitor AG1478 as well as the ERK inhibitor U0126, but not by the PI3K/PKB inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. CsA promoted the activation of ERK in JEG-3 cells, which was markedly abrogated in the presence of CXCL12 siRNA, or CXCR4 siRNA, or AG1478.

Conclusions

CsA may promote EGFR activation via CXCL12/CXCR4 axis, and EGFR downstream ERK signaling pathway may be involved in the CsA-induced proliferation of human trophoblast cells.