<Emphasis Type="Italic">Drosophila</Emphasis>-based <Emphasis Type="Italic">in vivo</Emphasis> assay for the validation of inhibitors of the epidermal growth factor receptor/Ras pathway

Overexpression of epidermal growth factor receptor (EGFR) is a common phenomenon observed in most cancers. Clinical treatment of such cancer involves the use of chemotherapeutic agents such as gefitinib and erlotinib which are inhibitors of tyrosine kinase (TK). These small molecules bind to the ATP-binding sites of the TK domain of EGFR. Our in silico analysis suggests that the TK domains of Drosophila and human EGFR are highly conserved. We therefore employed the Drosophila system to validate the in silico observations made with two important anticancer drugs. Since a large number of mutant flies are available, it was possible to investigate the various components of the EGFR/Ras/Raf/ MAPK pathways and the phosphorylation status of diphosphorylated extracellular signal-regulated kinase (dp-ERK1/2). These studies confirm the binding of the anilinoquinazolines to the Drosophila EGFR protein and modulation of its activity. Thus, Drosophila appears to be a robust and simple model system for screening newer anticancer drugs that act as TK inhibitors (TKIs). Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users. Supplementary figures and tables pertaining to this article are available on the Journal of Biosciences Website at http://www.ias.ac.in/jbiosci/dec2008/pp731-742-suppl.pdf     

Challenges in EGFRvIII Detection in Head and Neck Squamous Cell Carcinoma

ObjectiveHead and neck squamous cell carcinoma (HNSCC) accounts for more than 5% of all cancers worldwide. The mortality rate of HNSCC has remained unchanged (approximately 50%) over the last few decades. Ubiquitous overexpression of wild type EGFR in many solid tumors has led to the development of EGFR targeted therapies. EGFR can be constitutively activated via several mechanisms including the truncated, EGFR variant III isoform (EGFRvIII). EGFRvIII lacks exons 2–7 and has been reported to be present in up to 20–40% of HNSCC. EGFRvIII has been shown to contribute to cetuximab resistance. The mechanisms leading to EGFRvIII expression in HNSCC are unknown. The present investigation was undertaken to determine the etiology of EGFRvIII in HNSCC.ResultsUnlike glioma, EGFRvIII expression in HNSCC did not correlate with EGFR amplification. We found evidence of genomic deletion of the exon 2–7 in 6 of 7 HNSCC cases examined, however, the presence of genomic deletion did not always result in mRNA expression of EGFRvIII. RNA sequencing with automated alignment did not identify EGFRvIII due to microhomology between intron 1 and exon 8. RNA sequencing analyzed by manual alignment methods did not correlate well with RT-PCR and PCR findings.ConclusionThese findings suggest that genomic deletion as well as additional regulatory mechanisms may contribute to EGFRvIII expression in HNSCC. Further, large scale automated alignment of sequencing are unlikely to identify EGFRvIII and an assay specifically designed to detect EGFRvIII may be necessary to detect this altered form of EGFR in HNSCC tumors.     

Mutations and addiction to EGFR: the Achilles ‘heal’ of lung cancers?

The epidermal growth factor receptor (EGFR) gene product is a receptor tyrosine kinase (TK) that affects many important downstream pathways. The recent finding that mutations in EGFR predict the response of lung cancers to therapies that target the TK domain of the gene product has generated considerable interest. The mutations are associated with adenocarcinoma histology, oriental origin, female gender and never-smoker status. Most mutations target structures in the TK domain that appear to be essential for the phosphorylation function of the gene. Cancer cells with mutant EGFR genes might become physiologically dependent on the continued activity of the gene for the maintenance of their malignant phenotype; however, this might also be a target for therapy.     

Insight into binding mechanisms of EGFR allosteric inhibitors using molecular dynamics simulations and free energy calculations

Abstract

Lung cancer is the leading cause of cancer death, and epidermal growth factor receptor (EGFR) kinase domain mutations are a common cause of non-small-cell lung cancer (NSCLC), a major subtype of lung cancers. Patients harboring most of these mutations respond well to the EGFR inhibitors Gefitinib and Erlotinib initially, but soon develop resistance to them due to the emergence of the gatekeeper mutation T790M. The new-generation inhibitors such as AZD9291, HM61713, CO-1686 and WZ4002 can overcome T790M through covalent binding to Cys 797, but ultimately lose their efficacy upon the emergence of the C797S mutation that abolishes the covalent bonding. Allosteric inhibitors EAI001 and EAI045 are a new type of EGFR inhibitors that bind to EGFR away from the ATP-binding site and not relying on Cys 797. In this study, molecular dynamics simulations and free energy calculations were carried out on EAI001 and EAI045 in complex with EGFR, revealing the detailed inhibitory mechanism of EAI001 and EAI045 as EGFR allosteric inhibitor, which was expected to provide a basis for rational drug design of the EGFR allosteric inhibitors.

Communicated by Ramaswamy H. Sarma     

Prospective Evaluation of First-Line Erlotinib in Advanced Non-Small Cell Lung Cancer (NSCLC) Carrying an Activating EGFR Mutation: A Multicenter Academic Phase II Study in Caucasian Patients (FIELT)

Introduction

Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibition is the preferred first-line treatment of advanced adenocarcinoma of the lung that harbors EGFR activating tyrosine kinase domain mutations. Most data available pertain to Asian populations in which such mutations are more prevalent. We report on the long-term results of first-line treatment with erlotinib in Caucasian patients with advanced adenocarcinoma of the lung that have a somatic EGFR mutation in their tumor.

Methods

Multicenter academic prospective phase II study with erlotinib in patients with an activating EGFR tyrosine kinase (TK) domain somatic mutation (any exon encoding the kinase domain) in the tumor and no prior treatment for their advanced disease.

Results

Phenotypic preselecting of 229 patients led to a high EGFR mutation detection rate of 24% of which 46 patients were included in the phase II study. With a progression free survival (PFS) of 81% at three months the study met its primary endpoint for presumed superiority over chemotherapy. With an overall median PFS of 11 months and a median overall survival (OS) of 23 months, the results compare favorably with results obtained in randomized studies using TKI in first line in EGFR mutation positive adenocarcinoma of the lung.

Conclusion

The present study reinforces the use of EGFR tyrosine kinase inhibition (TKI) as a first line treatment of choice for advanced adenocarcinoma of the lung carrying an activating EGFR mutation. The mutation rate in preselected Caucasian patients is higher than previously reported. Issues relevant for clinical practice are discussed.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00339586","term_id":"NCT00339586"}}NCT00339586     

EGFR基因在非小细胞肺癌、乳腺癌中突变的研究

表皮生长因子受体(EGFR)基因酪氨酸激酶域体细胞突变与非小细胞肺癌(NSCLC)患者对酪氨酸激酶抑制剂吉非替尼敏感性密切相关。文章分析和检测本院75例非小细胞肺癌、10例乳腺癌患者石蜡包埋标本EGFR基因突变状况。采用PCR技术进行EGFR基因19和21外显子突变分析。结果显示:75例NSCLC患者中有13例(13/75,17.33%)酪氨酸激酶域存在体细胞突变。其中7例(7/75,9.33%)为19外显子缺失突变,6例(6/75,8%)为21外显子替代突变(2573T>G,L858R)。病理分型显示,腺癌突变率高于其他几种类型NSCLC。乳腺癌患者均为免疫组化HER-2阳性女性,EGFR基因的19、21外显子中未见突变发生。中国非小细胞肺癌患者总突变率高于高加索人种,女性患者较男性患者突变率高,提示肺腺癌的患者突变率高可能在吉非替尼的治疗中获益。     

Reactive oxygen species (ROS) reduce the expression of BRAK/CXCL14 in human head and neck squamous cell carcinoma cells

Abstract

The present study investigated the effects of oxidative stress induced by reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂) and hydroxyl radical (HO^?), on the expression of both BRAK , which is also known as non-ELR motif angiostatic CXC chemokine ligand 14 (CXCL14), in head and neck squamous cell carcinoma (HNSCC) cells. When HNSCC cells were cultured in the presence of ROS, the expression of BRAK was significantly decreased whereas that of IL-8 was increased. Interestingly, the effects on the expression of both genes in HNSCC cells were much greater with HO^? than with H₂O₂. The effects of ROS on both BRAK and IL-8 expression were attenuated by pre-treatment with N-acetyl-L-cysteine (NAC), epidermal growth factor receptor (EGFR), and mitogen-activated protein kinase (MAPK) inhibitors. These results indicate that oxidative stress induced by H₂O₂ or HO^? stimulates angiogenesis and tumuor progression by altering the gene expression of BRAK and IL-8 via the EGFR/MEK/ERK pathway in human HNSCC cells.     

Genetic variability of the coding region for the prion protein gene (PRNP) in gayal (Bos frontalis)

The gayal (Bos frontalis) is a rare semi-wild bovid species in which bovine spongiform encephalopathy (BSE) has not been reported. Polymorphisms of the prion protein gene (PRNP) have been correlated significantly with resistance to BSE. In this study, the coding region of PRNP was cloned and characterized in samples from 125 gayal. A total of ten single nucleotide polymorphisms (SNPs), including six silent mutations (C60T, G75A, A108T, G126A, C357T and C678T) and four mis-sense mutations (C8A, G145A, G461A and C756G), corresponding to amino acids T3K, G49S9, N154S and I252M were identified, revealing high genetic diversity. Three novel SNPs including C60T, G145A and C756G, which have not been reported previously in bovid species, were retrieved. There also was one insertion–deletion (187Del24) at the N-terminal octapeptide repeat region. Alignment of nucleotide and amino acid sequences showed a high degree of similarity with other bovid species. Using phylogenetic analyses it was revealed that gayal has a close genetic relationship with Zebu cattle. In short, preliminary information is provided about genotypes of the PRNP in gayal. This could assist with the study of the pathogenesis of transmissible spongiform encephalopathies and cross species transmission as well as a molecular breeding project for gayal in China.     

Protective effect of rosemary (Rosmarinus officinalis) against diethylnitrosamine-induced renal injury in rats

Abstract

Context: The kidney plays a central role in detoxification and excretion of toxic metabolites, and therefore, is susceptible to toxicity by xenobiotics.

Objective: To investigate the protective effect of Rosmarinus officinalis (rosemary) powder and its essential (volatile) oil against diethylnitrosamine (DEN)-induced renal injury in rats.

Materials and methods: Phenolic and flavonoid components were characterised in rosemary powder using HPLC-UV instrument while rosemary essential oil (E.O) was investigated via GC-MS technique. In rat model, rosemary was administrated orally (in diet) for two months. Lipid profile, antioxidant biomarkers, kidney functions and histopathological examinations were assessed.

Results: Hesperidin (4878.88?ppm) and ellagic acid (403.57?ppm) are among the major phenolic and flavonoid constituents in rosemary powder. Camphor (18.36%) and α-pinene (12.74%) represent the main E.O active ingredients. Rats treated with rosemary E.O showed a significant elevation in serum HDL (28.28%) accompanied by a decrease in LDL (115.47%). A significant decrease in serum creatinine and urea was also reported (69.72 and 109.89%, respectively). Moreover, serum glutathione peroxidise (GSH-Px) activity has been significantly increased. Kidney histopathological examinations confirmed the protective effect against DEN-induced abnormalities.

Conclusion: Rosemary (powder/E.O) was able to reduce or even prevent the severity of diethylnitrosamine-induced renal dysfunction.     

SLC4A11 mutations causative of congenital hereditary endothelial dystrophy (CHED) progressing to Harboyan syndrome in consanguineous Pakistani families

Background

Autosomal recessive corneal hereditary endothelial dystrophy (CHED) is a rare congenital disorder of cornea. Mutations in SLC4A11 gene are associated with CHED phenotype. CHED is also an early feature of Harboyan syndrome. The aim of the present study was to identify genetic mutations in the SLC4A11 gene in CHED cases belonging to inbred Pakistani families. Furthermore, all homozygous mutation carriers were investigated for hearing deficit.

Methods and results

This study included consanguineous CHED families presented at Al-Shifa Trust Eye Hospital, Rawalpindi, Pakistan from June 2018 to September 2018. DNA was extracted from blood samples. Direct sequencing of SLC4A11 gene was performed. All identified variants were evaluated by in silico programs i.e., SIFT, PolyPhen‐2, and MutationTaster. Pathogenicity of the two identified splice site variants was analyzed by Human Splicing Finder and MaxEnt Scan. Screening of five CHED families revealed a total of three previously un reported (p.Arg128Gly, c.2241-2A?>?T and c.1898-2A?>?C in family CHED19, CHED22 and CHED26 respectively) and two already reported homozygous disease causing variants (p.Arg869Cys and p.Val824Met in family CHED24 and CHED25 respectively) as predicted by mutation taster. All of these variants segregated with disease phenotype and were not detected in controls.

Conclusion

Affected individuals of the five CHED families screened in this study had the disease due to SLC4A11 mutations and progressing to Harboyan syndrome. Identification of previously unreported mutations aid to heterogeneity of SLC4A11 and CHED pathogenesis as well as helped to provide genetic counseling to affected families.

     

Prion protein gene sequence and chronic wasting disease susceptibility in white-tailed deer (Odocoileus virginianus)

ABSTRACT

The sequence of the prion protein gene (PRNP) affects susceptibility to spongiform encephalopathies, or prion diseases in many species. In white-tailed deer, both coding and non-coding single nucleotide polymorphisms have been identified in this gene that correlate to chronic wasting disease (CWD) susceptibility. Previous studies examined individual nucleotide or amino acid mutations; here we examine all nucleotide polymorphisms and their combined effects on CWD. A 626 bp region of PRNP was examined from 703 free-ranging white-tailed deer. Deer were sampled between 2002 and 2010 by hunter harvest or government culling in Illinois and Wisconsin. Fourteen variable nucleotide positions were identified (4 new and 10 previously reported). We identified 68 diplotypes comprised of 24 predicted haplotypes, with the most common diplotype occurring in 123 individuals. Diplotypes that were found exclusively among positive or negative animals were rare, each occurring in less than 1% of the deer studied. Only one haplotype (C, odds ratio 0.240) and 2 diplotypes (AC and BC, odds ratios of 0.161 and 0.108 respectively) has significant associations with CWD resistance. Each contains mutations (one synonymous nucleotide 555C/T and one nonsynonymous nucleotide 286G/A) at positions reported to be significantly associated with reduced CWD susceptibility. Results suggest that deer populations with higher frequencies of haplotype C or diplotypes AC and BC might have a reduced risk for CWD infection – while populations with lower frequencies may have higher risk for infection. Understanding the genetic basis of CWD has improved our ability to assess herd susceptibility and direct management efforts within CWD infected areas.     

Blood and sputum protein biomarkers for chronic obstructive pulmonary disease (COPD)

ABSTRACT

Introduction: Chronic obstructive pulmonary disease (COPD) is a heterogeneous set of disorders, characterized by airflow limitation, and reduced lung function. Despite increasing knowledge regarding its pathophysiology, there has been limited advancement in therapeutics and the current treatment strategy is symptom management and prevention of exacerbations.

Areas covered: Biomarkers represent important tools for the implementation of precision medicine. As fundamental molecules of all living processes, proteins could provide crucial information about how genes interact with the environment. Proteomics studies could act as important tools in identifying reliable biomarkers to enable a more precise therapeutic approach. In this review, we will explore the most promising blood and sputum protein biomarkers in COPD that have been consistently reported in the literature.

Expert commentary: Given the complexity of COPD, no single protein biomarker has been able to improve the outcomes of COPD patients. According to preliminary studies, precision medicine in COPD will likely require a combination of different proteins in a biomarker panel for clinical translation. With advancements in current mass spectrometry techniques, an enhancement in the identification of new biomarkers will be observed, and improvements in sequence database search can fill in potential gaps between biomarker discovery and patient care.     

Effect of genetic and environmental factors on protein biomarkers for common non-communicable disease and use of personally normalized plasma protein profiles (PNPPP)

Abstract

Objective: To study the impact of genetic and lifestyle factors on protein biomarkers and develop personally normalized plasma protein profiles (PNPPP) controlling for non-disease-related variance.

Materials and methods: Proximity extension assays were used to measure 145 proteins in 632 controls and 344 cases with non-communicable diseases.

Results: Genetic and lifestyle factors explained 20–88% of the variation in healthy controls. Adjusting for these factors reduced the number of candidate biomarkers by 63%.

Conclusion: PNPPP efficiently controls for non-disease-related variance, allowing both for efficient discovery of novel biomarkers and for covariate-independent linear cut-offs suitable for clinical use.     

Association of epidermal growth factor receptor (EGFR) gene polymorphism with lung cancer risk: a systematic review

Abstract

Epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family, which is thought to be involved in the development of cancer, as the EGFR gene is often amplified, and/or mutated in cancer cells. Lung cancer remains one of the most major causes of morbidity and mortality worldwide, accounting for more deaths than any other cancer cause. Gene polymorphism factor has been reported to be an important factor which increases the susceptibility of lung cancer. There lacks a well-documented diagnostic approach for the lung cancer risk, and the etiology of lung cancer is not clear. The current systematic review was performed to explore the association of EGFR gene polymorphism with lung cancer risk. In this review, association of EGFR 181946C?>?T, 8227G?>?A gene polymorphism with lung cancer was found, and EGFR Short genotype of cytosine adenine repeat number polymorphism was significantly associated with an increased risk of lung cancer.     

An In Vivo C. elegans Model System for Screening EGFR-Inhibiting Anti-Cancer Drugs

The epidermal growth factor receptor (EGFR) is a well-established target for cancer treatment. EGFR tyrosine kinase (TK) inhibitors, such as gefinitib and erlotinib, have been developed as anti-cancer drugs. Although non-small cell lung carcinoma with an activating EGFR mutation, L858R, responds well to gefinitib and erlotinib, tumors with a doubly mutated EGFR, T790M-L858R, acquire resistance to these drugs. The C. elegans EGFR homolog LET-23 and its downstream signaling pathway have been studied extensively to provide insight into regulatory mechanisms conserved from C. elegans to humans. To develop an in vivo screening system for potential cancer drugs targeting specific EGFR mutants, we expressed three LET-23 chimeras in which the TK domain was replaced with either the human wild-type TK domain (LET-23::hEGFR-TK), a TK domain with the L858R mutation (LET-23::hEGFR-TK[L858R]), or a TK domain with the T790M-L858R mutations (LET-23::hEGFR-TK[T790M-L858R]) in C. elegans vulval cells using the let-23 promoter. The wild-type hEGFR-TK chimeric protein rescued the let-23 mutant phenotype, and the activating mutant hEGFR-TK chimeras induced a multivulva (Muv) phenotype in a wild-type C. elegans background. The anti-cancer drugs gefitinib and erlotinib suppressed the Muv phenotype in LET-23::hEGFR-TK[L858R]-expressing transgenic animals, but not in LET-23::hEGFR-TK[T790M-L858R] transgenic animals. As a pilot screen, 8,960 small chemicals were tested for Muv suppression, and AG1478 (an EGFR-TK inhibitor) and U0126 (a MEK inhibitor) were identified as potential inhibitors of EGFR-mediated biological function. In conclusion, transgenic C. elegans expressing chimeric LET-23::hEGFR-TK proteins are a model system that can be used in mutation-specific screens for new anti-cancer drugs.     

Intracellular pH regulation in ventral horn neurones cultured from embryonic rat spinal cord

Summary

We have isolated and characterized a cDNA from the marine sponge Geodia cydonlum coding for a new member of the tyrosine protein kinase (TK) family. The cDNA encodes a protein of M_r = 68 710, termed GCTK, which is homologous to class II receptor tyrosine kinases (RTKs). GCTK contains conserved amino acids (aa) characteristic of all protein kinases, and the sequences DLATRN and PIRWMATE which are highly specific for TKs. Furthermore, the sequence N-L-Y-x(3)-Y-Y-R Is highly homologous to the sequence D-[LIV]-Y-x(3)-Y-Y-R found only in class II RTKs. The sponge TK, when compared with mammalian class II RTKs, shows maximum 31% homology in the TK domain indicating that this the oldest member of class II RTK started to diverge from the common ancestral protein kinase 650 million years ago. Using GCTK as a probe we identified three mRNA signals ranging from 2μ6 to 0μ6 kb. Kinase activity was localized only in the cell membranes from G. cydonium (M_r = 65 000), and was not detected in the cytosol of this organism. Antibodies raised against a synthetic peptide, corresponding to the aa residues within the catalytic domain of the sponge TK, recognized strongly two proteins of M_r = 65 000; these proteins, present in membrane fractions, also bound to the anti-phosphotyrosine antibody. These data suggest that the TK cloned from the sponge is a membrane-associated 65 kDa protein. Moreover these results demonstrate that RTKs are present from the lowest group of multicellular eukaryotes, sponges, to mammals, and may suggest that RTKs are involved in a signal transduction pathway.     

Functional polymorphisms of caveolin-1 variants as potential biomarkers of esophageal squamous cell carcinoma

Abstract

Objective: To investigate the association of caveolin-1 (CAV1) genetic variants (C239A (rs1997623), G14713A (rs3807987), G21985A (rs12672038), T29107A (rs7804372)) with esophageal squamous cell carcinoma (ESCC) susceptibility.

Methods: A total of 427 patients with ESCC and 427 healthy controls were genotyped using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method.

Results: There were significant differences between patients and controls in distributions of their genotypes and allelic frequencies in G14713A and T29107A polymorphisms. Furthermore, haplotype analysis revealed that haplotypes CAAT and CAGT were associated with high risk for ESCC, while haplotype CGGA was protective against ESCC. Stratified analysis showed the associations between the SNPs (G14713A and T29107A) and ESCC risk were noteworthy among female patients and patients who never smoke or drank alcohol.

Conclusions: Genetic polymorphisms of CAV1 G14713A and T29107A might affect an individual’s susceptibility in developing ESCC, making them efficient potential genetic biomarkers for early detection of ESCC.     

Potential antitumour and pro-oxidative effects of (E)-methyl 2-(7-chloroquinolin-4-ylthio)-3-(4-hydroxyphenyl) acrylate (QNACR)

Context: Characterization of the pro-oxidant activity of QNACR.

Objectives: Reactive oxygen species (ROS) induce cellular damage and represent unique opportunities to kill malignant cells. In this study, we synthesized and evaluated the new compound, (E)-methyl 2-(7-chloroquinolin-4-ylthio)-3-(4-hydroxyphenyl) acrylate (QNACR) as potential pro-oxidative agent against breast cancer.

Methods: Oxidative stress biomarkers such as ROS, thiobarbuturic acid reactive species (TBARs) and different antioxidant enzyme activities were determined in cell lysates.

Results: QNACR showed cytotoxic and more selective effects to tumour MCF7 cells (IC₅₀ < 25 µM) compared to antitumour controls, inducing ROS and TBARs parallel to inhibitions of catalase (CAT), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Longer exposures to QNACR triggered adaptive effects increasing the overall activities of CAT, glutathione reductase, G6PDH and 6PGDH, but eventually the adaptation changes faded and cells died.

Conclusion: QNACR led to remarkable modifications in the oxidative status of tumour cells, proposing this compound as potential alternative for antitumour therapy.