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Gene Therapy for Small Cell Lung Cancer
- Targeting the Neuroendocrine Phenotype


Thomas Tuxen Poulsen  


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Accepted by: Faculty of Health University of Copenhagen
Defended on: October 3, 2008
Official opponents: Professor Finn Cilius Nielsen (Chair) , Professor Thomas G. Jensen , Chief Oncologist Olfred Hansen
Tutors: Associate Professor Hans Skovgaard Poulsen , Senior Scientist Nina Pedersen , Principal Investigator Ilona R. Linnoila

Published in the PhD Database: October 6, 2008


English abstract
Small cell lung cancer (SCLC) is a highly aggressive smoking-associated malignancy with poor prognosis, provding an urgent need for the development of novel systemic treatments. SCLC cells exhibit high expression of numerous developmental neuroendocrine markers, some of which are absent or expressed at trace levels in adult normal tissues. Many of these markers are suspected to play central roles in the unresolved transition from normal cell to SCLC.
The primary focus of the current PhD-project has been to develop a novel transcriptionally targeted gene therapy for SCLC-treatment. The strategy is based on the use of regulatory regions from one or more SCLC specific gene(s) to drive the expression of a cytotoxic suicide gene in order to specifically induce cell death in SCLC cells. By Microarray profiling we identified SCLC specific genes with potential promoter candidates for this purpose. In vitro studies confirmed that regulatory regions from the human Achaete Scute Homolog 1 and Enhancer of Zeste Homolog 2 genes potently activate reporter and therapeutic gene expression in SCLC cells. Furthermore, a chimeric construct comprising regions from both promoters induces high gene expression exclusively in SCLC cells while retaining specificity in control cells of different origin.
In a search for surface receptor candidates for targeting gene therapy to SCLC cells, the Neuronal Pentraxin Receptor was identified. Because previous reports identified Neuronal Pentraxins as binding partners for the snake venom neurotoxin taipoxin, the toxic effect of taipoxin on SCLC cell survival was investigated. The neurotoxin was found to potently induce cell death at nanomolar drug concentrations to which control cells remained insensitive.
Finally, to gain insight into the neuroendocrine transitions in the lung epithelium occuring during exposure to tobacco related carcinogens; expression of selected neuroendocrine (and SCLC) markers was investigated in mice after exposure to the carcinogen naphthalene. Further knowledge in this field could potentially aid the understanding of the processes involved in lung carcinogenesis. On the basis of immunohistochemical and quantitative RT-PCR studies, increased expression of the neuroendocrine marker Protein Gene Product 9.5 (PGP9.5) was observed in the non-neuroendocrine pulmonary epithelium, potentially pointing to a role for this protein in early lung carcinogenesis.



Danish abstract
Småcellet lungecancer (SCLC) er en særdeles ondartet kræftform, der er tæt associeret til tobaksrygning. De eksisterende behandlingstilbud er utilstrækkelige, hvilket giver sig udslag i en dårlig prognose og der er et stort behov for udvikling af nye systemiske behandlinger. SCLC celler udtrykker et udvalg af neuroendokrine markører og enkelte af disse er ikke udtrykt i de normale væv hos voksne. Mange af disse markører mistænkes at spille en central rolle for transitionen af normal celle til SCLC ¿ en proces, der langt fra er kortlagt.
Det primære mål med dette PhD-studium har været at udvikle en transkriptionelt reguleret genterapeutisk strategi til behandling af SCLC. Strategien går ud på at afprøve regulatoriske regioner fra et eller flere gener, der er specifikt udtrykt i SCLC, som aktivatorer af et cytotoksisk gen. Formålet er at opnå en regulering, der tillader at genet kun aktiveres i SCLC cellerne og slår disse ihjel, mens kroppens normale væv forbliver upåvirkede af behandlingen. Ved hjælp af Microarray-analyse af genekspressionsprofilen i SCLC har vi identificeret et udvalg af gener med SCLC specifik ekspression og regulatoriske regioner fra to af disse: Achaete-Scute Homolog 1 og Enhancer of Zeste Homolog 2 viste sig at være særdeles aktive i SCLC celler. Derudover var et fusionskontrukt bestående af udvalgte regulatoriske regioner fra begge disse gener i stand til at inducere SCLC-specifik gen-ekspression og celledød.
Samme Microarray analyse viste højt og specifikt udtryk af den Neuronale Pentraxin Receptor i SCLC. Neuronale Pentraxiner er tidligere blevet associeret med binding til slangegift-toksinet taipoxin i neuroner og dette førte til undersøgelse af effekten af taipoxin i SCLC-celler. Taipoxin viste sig at være cytotoksisk i SCLC ved koncentrationer, der ikke var toksiske for en række kontrolceller.
Idet en øget forståelse for de neuroendokrine ændringer, der opstår i lungeepithelet efter tobaksrøgseksponering formentlig vil kunne bidrage til at forklare ætiologien bag SCLC, blev udtrykket af forskellige neuroendokrine markører undersøgt i lunger på mus eksponeret for carcinogenet naphthalen, der findes i tobaksrøg. Ved hjælp af immunhistokemi og kvantitativ RT-PCR detekteredes en midlertidig stigning i ekspressionen af den neuroendokrine markør PGP9.5, efter naphthalen-behandling, hvilket indikerer at dette protein har betydning for tidlig lungecarcinogenese.