A JAK-STAT pathway regulates wing vein formation in Drosophila.

We present evidence that the JAK-STAT signal transduction pathway regulates multiple developmental processes in Drosophila. We screened for second-site mutations that suppress the phenotype of the hyperactive hopTum-1 Jak kinase, and recovered a mutation that meiotically maps to the known chromosomal position of D-Stat, a Drosophila stat gene. This hypomorphic mutation, termed statHJ contains a nucleotide substitution in the first D-Stat intron, resulting in a reduction in the number of correctly processed transcripts. Further, the abnormally processed mRNA encodes a truncated protein that has a dominant negative effect on transcriptional activation by the wild-type cDNA in cell culture. statHJ mutants exhibit patterning defects that include the formation of ectopic wing veins, similar to those seen in mutants of the epidermal growth factor/receptor pathway. Abnormalities in embryonic and adult segmentation and in tracheal development were also observed. The hopTum-1 and statHJ mutations can partially compensate for each other genetically, and Hop overexpression can increase D-Stat transcriptional activity in vitro, indicating that the gene products interact in a common regulatory pathway.

A tyrosine kinase profile of prostate carcinoma.

Tyrosine kinases play central roles in the growth and differentiation of normal and tumor cells. In this study, we have analyzed the general tyrosine kinase expression profile of a prostate carcinoma (PCA) xenograft, CWR22. We describe here an improved reverse transcriptase-PCR approach that permits identification of nearly 40 different kinases in a single screening; several of these kinases are newly cloned kinases and some are novel. According to this, there are 11 receptor kinases, 9 nonreceptor kinases, and at least 7 dual kinases expressed in the xenograft tissue. The receptor kinases include erbB2, erbB3, Ret, platelet-derived growth factor receptor, sky, nyk, eph, htk, sek (eph), ddr, and tkt. The nonreceptor kinases are lck, yes, abl, arg, JakI, tyk2, and etk/bmx. Most of the dual kinases are in the mitogen-activating protein (MAP) kinase-kinase (MKK) family, which includes MKK3, MKK4, MEK5, and a novel one. As a complementary approach, we also analyzed by specific reverse transcriptase-PCR primers the expression profile of erbB/epidermal growth factor receptor family receptors in a variety of PCA specimens, cell lines, and benign prostatic hyperplasia. We found that erbB1, -2, and -3 are often coexpressed in prostate tissues, but not in erbB4. The information established here should provide a base line to study the possible growth and oncogenic signals of PCA.

Ligand-directed retroviral targeting of human breast cancer cells.

We explored the feasibility of designing retroviral vectors that can target human breast cancer cells with characteristic receptors via ligand-receptor interaction. The ecotropic Moloney murine leukemia virus envelope was modified by insertion of sequences encoding human heregulin. Ecotropic virus, which normally does not infect human cells, when pseudotyped with the modified envelope protein now crosses species to infect human breast cancer cell lines that overexpress HER-2 (human epidermal growth factor receptor; also called ERBB2) and HER-4 (also called ERBB4), while human breast cancer cell lines expressing low levels of these receptors remain resistant to infection. Since about 20% of human breast cancers overexpress HER-2 and some of breast cancer cell lines overexpress both HER-2 and HER-4, cell-specific targeting of retroviral vectors may provide a different approach for in vivo gene therapy of this type of breast cancer.

Identificazione di miRNA correlati ad EGFR: nuovi potenziali biomarcatori di risposta alla terapia di 2a-3a linea in pazienti con NSCLC metastatico

La diagnosi di neoplasia epiteliale maligna polmonare è legata tradizionalmente alla distinzione tra carcinoma a piccole cellule (small-cell lung cancer, SCLC) e carcinoma non-a piccole cellule del polmone (non-small-cell lung cancer, NSCLC). Nell’ambito del NSCLC attualmente è importante di-stinguere l’esatto istotipo (adenocarcinoma, carcinoma squamocellulare e carcinoma neuroendocrino) perchè l’approccio terapeutico cambia a seconda dell’istotipo del tumore e la chemioterapia si dimostra molto spesso inefficace. Attualmente alcuni nuovi farmaci a bersaglio molecolare per il gene EGFR, come Erlotinib e Gefitinib, sono utilizzati per i pazienti refrattari al trattamento chemioterapico tradizionale, che non hanno risposto a uno o più cicli di chemioterapia o che siano progrediti dopo questa. I test per la rilevazione di specifiche mutazioni nel gene EGFR permettono di utilizzare al meglio questi nuovi farmaci, applicandoli anche nella prima linea di trattamento sui pazienti che hanno una maggiore probabilità di risposta alla terapia. Sfortunatamente, non tutti i pazienti rispondono allo stesso modo quando trattati con farmaci anti-EGFR. Di conseguenza, l'individuazione di biomarcatori predittivi di risposta alla terapia sarebbe di notevole importanza per aumentare l'efficacia dei questi farmaci a target molecolare e trattare con farmaci diversi i pazienti che con elevata probabilità non risponderebbero ad essi. I miRNAs sono piccole molecole di RNA endogene, a singolo filamento di 20-22 nucleotidi che svolgono diverse funzioni, una delle più importanti è la regolazione dell’espressione genica. I miRNAs possono determinare una repressione dell'espressione genica in due modi: 1-legandosi a sequenze target di mRNA, causando così un silenziamento del gene (mancata traduzione in proteina), 2- causando la degradazione dello specifico mRNA. Lo scopo della ricerca era di individuare biomarcatori capaci di identificare precocemente i soggetti in grado di rispondere alla terapia con Erlotinib, aumentando così l'efficacia del farmaco ed evitan-do/riducendo possibili fenomeni di tossicità e il trattamento di pazienti che probabilmente non ri-sponderebbero alla terapia offrendo loro altre opzioni prima possibile. In particolare, il lavoro si è fo-calizzato sul determinare se esistesse una correlazione tra la risposta all'Erlotinib ed i livelli di espressione di miRNAs coinvolti nella via di segnalazione di EGFR in campioni di NSCLC prima dell’inizio della terapia. Sono stati identificati 7 microRNA coinvolti nel pathway di EGFR: miR-7, -21, 128b, 133a, -133b, 146a, 146b. Sono stati analizzati i livelli di espressione dei miRNA mediante Real-Time q-PCR in campioni di NSCLC in una coorte di pazienti con NSCLC metastatico trattati con Erlotinib dal 1° gennaio 2009 al 31 dicembre 2014 in 2°-3° linea dopo fallimento di almeno un ciclo di chemioterapia. I pazienti sottoposti a trattamento con erlotinib per almeno 6 mesi senza presentare progressione alla malattia sono stati definiti “responders” (n=8), gli altri “non-responders” (n=25). I risultati hanno mostrato che miR-7, -133b e -146a potrebbero essere coinvolti nella risposta al trat-tamento con Erlotinib. Le indagini funzionali sono state quindi concentrate su miR-133b, che ha mo-strato la maggiore espressione differenziale tra i due gruppi di pazienti. E 'stata quindi studiata la capacità di miR-133b di regolare l'espressione di EGFR in due linee di cellule del cancro del polmone (A549 e H1299). Sono stati determinati gli effetti di miR-133b sulla crescita cellulare. E’ stato anche analizzato il rapporto tra miR-133b e sensibilità a Erlotinib nelle cellule NSCLC. L'aumento di espressione di miR-133b ha portato ad una down-regolazione del recettore di EGF e del pathway di EGFR relativo alla linea cellulare A549. La linea cellulare H1299 era meno sensibili al miR-133b up-regulation, probabilmente a causa dell'esistenza di possibili meccanismi di resistenza e/o di com-pensazione. La combinazione di miR-133b ed Erlotinib ha aumentato l'efficacia del trattamento solo nella linea cellulare A549. Nel complesso, questi risultati indicano che miR-133b potrebbe aumentare / ripristinare la sensibilità di Erlotinib in una frazione di pazienti.

Rhomboid intramembrane protease RHBDL4 triggers ER-export and non-canonical secretion of membrane-anchored TGFα

Rhomboid intramembrane proteases are the enzymes that release active epidermal growth factor receptor (EGFR) ligands in Drosophila and C. elegans, but little is known about their functions in mammals. Here we show that the mammalian rhomboid protease RHBDL4 (also known as Rhbdd1) promotes trafficking of several membrane proteins, including the EGFR ligand TGFα, from the endoplasmic reticulum (ER) to the Golgi apparatus, thereby triggering their secretion by extracellular microvesicles. Our data also demonstrate that RHBDL4-dependent trafficking control is regulated by G-protein coupled receptors, suggesting a role for this rhomboid protease in pathological conditions, including EGFR signaling. We propose that RHBDL4 reorganizes trafficking events within the early secretory pathway in response to GPCR signaling. Our work identifies RHBDL4 as a rheostat that tunes secretion dynamics and abundance of specific membrane protein cargoes.

Polymerizable Peptide Monomers for the Targeted and Intracellular Delivery of Cancer Therapeutics

Thesis (Ph.D.)--University of Washington, 2016-06

A small-molecule lead compound for the treatment of Alzheimer's disease

At autopsy, Alzheimer's disease is characterised by the presence of amyloid plaques and neurofibrillary tangles, made up of two peptide sequences, amyloid-beta(1-40) (A beta 40) and amyloid-beta(1-42) (A beta 42). In Tyrode's solution (2 mM Ca2+), 10 mu M A beta 42 peptide almost immediately aggregates and eventually forms p-sheets. This aggregation can be inhibited with 4,5-dianilinophthalimide (DAPH). Ca2+-permeant AMPA receptors are involved in the neuronal Ca2+ influx (neurotoxicity) induced by the A beta 42 peptide in cultured neuronal cells. The Ca2+ influx observed with pre-incubated A beta 42 peptide was inhibited by DAPH. DAPH also inhibits epidermal growth factor receptor kinase, and this will prevent its development for use in Alzheimer's disease. The potential of DAPH as a small-molecule lead compound for the treatment of Alzheimer's disease next requires the separation of the structural requirements that reverse fibril formation and inhibit epidermal growth factor receptor kinase.

Distinct types of glial cells populate the Drosophila antenna

Background: The development of nervous systems involves reciprocal interactions between neurons and glia. In the Drosophila olfactory system, peripheral glial cells arise from sensory lineages specified by the basic helix- loop- helix transcription factor, Atonal. These glia wrap around the developing olfactory axons early during development and pattern the three distinct fascicles as they exit the antenna. In the moth Manduca sexta, an additional set of central glia migrate to the base of the antennal nerve where axons sort to their glomerular targets. In this work, we have investigated whether similar types of cells exist in the Drosophila antenna. Results: We have used different P( Gal4) lines to drive Green Fluorescent Protein ( GFP) in distinct populations of cells within the Drosophila antenna. Mz317:: GFP, a marker for cell body and perineural glia, labels the majority of peripheral glia. An additional similar to 30 glial cells detected by GH146:: GFP do not derive from any of the sensory lineages and appear to migrate into the antenna from the brain. Their appearance in the third antennal segment is regulated by normal function of the Epidermal Growth Factor receptor and small GTPases. We denote these distinct populations of cells as Mz317- glia and GH146- glia respectively. In the adult, processes of GH146- glial cells ensheath the olfactory receptor neurons directly, while those of the Mz317- glia form a peripheral layer. Ablation of GH146- glia does not result in any significant effects on the patterning of the olfactory receptor axons. Conclusion: We have demonstrated the presence of at least two distinct populations of glial cells within the Drosophila antenna. GH146- glial cells originate in the brain and migrate to the antenna along the newly formed olfactory axons. The number of cells populating the third segment of the antenna is regulated by signaling through the Epidermal Growth Factor receptor. These glia share several features of the sorting zone cells described in Manduca.

EGFR amplification and lack of activating mutations in metaplastic breast carcinomas

Metaplastic breast carcinomas are reported to harbour epidermal growth factor receptor (EGFR) overexpression in up to 80% of the cases, but EGFR gene amplification is the underlying genetic mechanism in around one-third of these. In this study, EGFR gene amplification as defined by chromogenic in situ hybridization and protein overexpression was examined in a cohort of 47 metaplastic breast carcinomas. Furthermore, the presence of activating EGFR mutations in exons 18, 19, 20, and 21 was investigated. Thirty-two cases showed EGFR overexpression and of these, 11 (34%) harboured EGFR gene amplification. In addition, EGFR amplification showed a statistically significant association with EGFR overexpression (p < 0.0094) and was restricted to carcinomas with homologous metaplasia. Ten cases, five with and five without EGFR amplification, were subjected to microarray-based CGH, which demonstrated that EGFR copy number gain may occur by amplification of a discrete genomic region or by gains of the short arm of chromosome 7 with a breakpoint near the EGFR gene locus, the minimal region of amplification mapping to EGFR, LANCL2, and SECOG. No activating EGFR mutations were identified, suggesting that this is unlikely to be a common alternative underlying genetic mechanism for EGFR expression in metaplastic breast carcinomas. Given that metaplastic breast carcinomas are resistant to conventional chemotherapy or hormone therapy regimens and that tumours with EGFR amplification are reported to be sensitive to EGFR tyrosine kinase inhibitors, these findings indicate that further studies are warranted to explore EGFR tyrosine kinase inhibitors as potential therapeutic agents for metaplastic breast carcinomas harbouring amplification of 7p11.2. Copyright (c) 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Functional proteomics to dissect tyrosine kinase signalling pathways in cancer

Advances in the generation and interpretation of proteomics data have spurred a transition from focusing on protein identification to functional analysis. Here we review recent proteomics results that have elucidated new aspects of the roles and regulation of signal transduction pathways in cancer using the epidermal growth factor receptor (EGFR), ERK and breakpoint cluster region (BCR)-ABL1 networks as examples. The emerging theme is to understand cancer signalling as networks of multiprotein machines which process information in a highly dynamic environment that is shaped by changing protein interactions and post-translational modifications (PTMs). Cancerous genetic mutations derange these protein networks in complex ways that are tractable by proteomics.

Cellular uptake and biological activity of synthetic hammerhead ribozymes

Glioblastoma Multiforme (GBM) is a highly malignant form of brain cancer for which there is no effective cure. The over-expression of a number of genes, including the epidermal growth factor receptor (EGFr), has been implicated as a causative factor of tumourigenesis. Ribozymes are a class of ribonucleic acid that possess enzymatic properties. They can inhibit gene-expression in a highly sequence specific manner by catalysing the trans-cleavage of target RNA. The potential use of synthetic hammerhead ribozymes as novel anti-brain tumour agents was investigated in this study. The successful use of synthetic, exogenously administered ribozymes for such applications will require chemical modifications that improve biological stability and a fundamental understanding of cellular uptake mechanisms. Chimeric 2'-O-methylated hammerhead ribozymes proved to be significantly more stable (>4000-fold) in serum than unmodified RNA ribozymes and exhibited high in vitro catalytic activity. The cellular association of an internally [32P]-labelled 2'-O-methylated chimeric ribozyme in U87-MG human glioma cells was temperature-, energy- and pH-dependent and involved an active process that could be competed with a variety of polyanions. Indications are that the predominant mechanism of uptake is by adsorptive and / or receptor mediated endocytosis. Twenty 2'-O-methylated chimeric ribozymes were designed to cleave various sites along the EGFr mRNA. In vitro, 18 ribozymes exhibited high activity in cleaving a complementary short substrate. Using LipofectAMINETM as a delivery agent, the efficacy of these ribozymes was evaluated in the A431 cell line, which expresses amplified levels of EGFr. Studies revealed that although the ribozymes were taken up by the cells and remained stable over a period of 4 days, no significant reduction in either EGFr expression or cell proliferation was evident. The presence of telomerase, a ribonucleoprotein responsible for telomere elongation, has been strongly associated with tumour progression. The biological activity of a 2'-O-methylated ribozyme targeted against the RNA component of telomerase was determined. The ribozyme exhibited specific dose-dependent inhibition of telomerase activity in U87-MG cell lysates with an IC50 of –4μM. When 4μM ribozyme was delivered to intact U87-MG cells, complexed to LipofectAMINETM, telomerase activity was significantly reduced to 74.5±4.17% of the untreated control. Free ribozyme showed no significant inhibitory effect demonstrating the importance of an appropriate delivery system for optimum delivery of exogenously administered ribozymes.

DNA chip designed antisense oligodeoxynucleotides targeting EGFR MRNA for brain tumour therapy

Glioblastoma multiforme (GBM) is a malignant brain tumour for which there is currently no effective treatment regime. It is thought to develop due to the overexpression of a number of genes, including the epidermal growth factor receptor (EGFR), which is found in over 40% of GBM. Novel forms of treatment such as antisense therapy may allow for the specific inhibition of aberrant genes and thus they are optimistic therapies for future treatment of GBM. Oligodeoxynucleotides (ODNs) are small pieces of DNA that are often modified to increase their stability to nucleases and can be targeted to the aberrant gene in order to inhibit it and thus prevent its transcription into protein. By specifically binding to mRNA in an antisense manner, they can bring about its degradation by a variety of mechanisms including the activation of RNase H and thus have great potential as therapeutic agents. One of the main drawbacks to the utilisation of this therapy so far is the lack of techniques that can successfully predict accessible regions on the target mRNA that the ODNs can bind to. DNA chip technology has been utilised here to predict target sequences on the EGFR mRNA and these ODNs (AS 1 and AS2) have been tested in vitro for their stability, uptake into cells and their efficacy on cellular growth, EGFR protein and mRNA. Studies showed that phosphorothioate and 2'O-methyl ODNs were significantly more stable than phosphodiester ODNs both in serum and serum-free conditions and that the mechanism of uptake into A431 cells was temperature dependent and more efficient with the use of optimised lipofectin. Efficacy results show that AS 1 and AS2 phosphorothioate antisense ODNs were capable of inhibiting cell proliferation by 69% ±4% and 65% ±4.5% respectively at 500nM in conjunction with a non-toxic dose of lipofectinTM used to enhance cellular delivery. Furthermore, control ODN sequences, 2' O-methyl derivatives and a third ODN sequence, that was found not to be capable of binding efficiently to the EGFR mRNA by DNA chip technology, showed no significant effect on cell proliferation. AS 1 almost completely inhibited EGFR protein levels within 48 hours with two doses of 500nM AS 1 with no effect on other EGFR family member proteins or by control sequences. RNA analysis showed a decrease in mRNA levels of 32.4% ±0.8% but techniques require further optimisation to confirm this. As there are variations found between human glioblastoma in situ and those developed as xenografts, analysis of effect of AS 1 and AS2 was performed on primary tumour cell lines derived from glioma patients. ODN treatment showed a specific knockdown of cell growth compared to any of the controls used. Furthermore, combination therapies were tested on A431 cell growth to determine the advantage of combining different antisense approaches and that of conventional drugs. Results varied between the combination treatments but indicated that with optimisation of treatment regimes and delivery techniques that combination therapies utilising antisense therapies would be plausible.

Cellular delivery of hammerhead ribozymes

Hammerhead ribozymes are potent RNA molecules which have the potential to specifically inhibit gene expression by catalysing the trans-cleavage of mRNAs. However, they are unstable in biological fluids and cellular delivery poses a problem. Site-specific chemical modification of hammerhead ribozymes was evaluated as a means of enhancing biological stability. Chimeric, 2'-O-methylated ribozymes, containing only five unmodified ribonucleotides, were catalytically active in vitro (kcat = 1.46 min-1) and were significantly more stable in serum and lysosomal enzymes than unmodified (all-RNA) counterparts. Furthermore, they remained undegraded in cell-containing media for up to 8 hours. Stability enhancement allowed cellular uptake properties of radiolabelled ribozymes to be assessed following exogenous delivery. Studies in vulval and glial cell lines indicated that chimeric ribozymes became cell-associated via an inefficient process, which was energy and concentration dependant. A considerable proportion of ribozymes remained bound to cell-surface components, however, a small proportion (<1%) were internalised via mechanisms of adsorptive and / or receptor mediated endocytosis. Fluorescent microscopy indicated that ribozymes were localised within endosomal / lysosomal vesicles following cell entry. This was confirmed by immuno-electron microscopy, which allowed the detection of biotin-labelled ribozymes within the cell ultrastructure. Despite the predominant localisation within endocytic vesicles, a small proportion of internalised ribozymes appeared able to exit these compartments and penetrate target sites within the nucleus and cytoplasm. The ribozymes designed in this report were directed against the epidermal growth factor receptor mRNA, which is over-expressed in a malignant brain disease called glioblastoma multiforme. In order to examine the fate of ribozymes in the brain, the distribution of FITC-labelled ribozymes was examined following intra-cerebro ventricular injection to mice. FITC-ribozymes demonstrated high punctate pattern of distribution within the striatum and cortex, which appeared to represent localisation within cell bodies and dendritic processes. This suggested that delivery to glial cells in vivo may be possible. Finally, strategies were investigated to enhance the cellular delivery of ribozymes. Conjugation of ribozymes to anti~transferrin receptor antibodies improved cellular uptake 3-fold as a result of a specific interaction with transferrin receptors. Complexation with cationic liposomes also significantly improved cell association, however, some toxiclty was observed and this could be a limitation to their use. Overall, it would appear that hammerhead ribozymes can be chemically stabilised to allow direct exogenous administration in vivo. However, additional delivery strategies are probably required to improve cellular uptake, and thus, allow ribozymes to achieve their full potential as pharmaceutical agents. KEYWORDS: Catalytic

Synthesis and biological properties of 2-phenylbenzothiazole derivatives

2-Phenylbenzothiazoles have structural similarities to the antioestrogenic 2-phenylindole, zindoxifene and to the oestrogenic isoflavone, genistein which also inhibits tyrosine kinases. Hydroxylated 2-phenylbenzothiazole derivatives were therefore produced and tested for oestrogenic and tyrosine kinase inhibitory activity. Synthesis of methoxy substituted 2-phenylbenzothiazoles was via the Jacobson method, demethylation being effected by boron tribromide at -70oC. Three amino substituted 2-phenylbenzothiazoles were also synthesised and tested for activity. Data is presented for oestrogen receptor binding activity, aromatase inhibitory activity, epidermal growth factor receptor tyrosine kinase (EGFRTK) inhibitory activity and cytotoxicity to ANN-1, 3T3, MCF-7 and WIDR cells. Oestrogen receptor binding affinity (RBA) was shown by five of the nine compounds tested. 2-(4-hydroxy)-6-hydroxybenzo-thiazole was the most active of the benzothiazoles tested (RBA 0.7). This is low but comparable to that of genistein. EGFRTK inhibitory activity was shown by four of the six benzothiazole derivatives tested; activity was comparable to that of genistein. Cytotoxicity assays have shown no selective toxicity of 2-phenylbenzothiazoles to any of the cell lines tested. Toxicity to MCF-7 cells was similar to that for other cell lines despite some compounds showing oestrogen receptor binding capacity. Amino-substituted 2-phenylbenzothiazoles showed selective toxicity towards transformed ANN-1 cells compared to normal 3T3 cells but the mechanism of this selectivity has not been established. Molecular modelling techniques, including CHEM-X, QUANTA and MOPAC were used to compare known ATP-competitive tyrosine kinase inhibitors with a model of ATP built from the crystal structure of the ATP-phosphoglycerate kinase complex. Structural features thought to be important to kinase inhibition were found and used to suggest further 2-phenylbenzothiazole analogues which may have improved activity.