The role of INK4a/Arf and EGFR genes in the development of glioblastoma

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Serval ID
serval:BIB_42733
Type
PhD thesis: a PhD thesis.
Collection
Publications
Institution
Title
The role of INK4a/Arf and EGFR genes in the development of glioblastoma
Author(s)
Lachat Y.
Director(s)
Hegi M.
Institution details
Université de Lausanne, Faculté de biologie et médecine
Address
Lausanne
Publication state
Accepted
Issued date
2004
Language
english
Number of pages
106
Notes
REROID:R003641834; 30 cm ill.; Old school value: Université de Lausanne
Abstract
Résumé
Le glioblastome multiforme est la forme tumorale la plus fréquente et la plus maligne du système nerveux chez l'homme. La survie des patients atteints de glioblastome est très faible et n'excède que rarement une année, malgré tous les efforts dans le domaine de la chirurgie, chimiothérapie et radiothérapie. L'amplification du gène egfr et l'expression de son mutant tronqué (ΔEGFR(2-7)) associés à la perte du gène INK4a/Arf sont fréquentes dans le glioblastome primaire. Ainsi, l'activation d'egfr et la perte du gène INK4/Arf sont supposés avoir un effet coopératif dans le développement du glioblastome.
Cette notion de coopération a été étudiée dans un système faisant appel à la transplantation de cellules neuroectodermales exprimant ΔEGFR(2-7) et déficientes pour le gène INK4a/Arf, dans le cerveau de souris. Ce modèle animal a permis d'induire le développement de glioblastomes, démontrant ainsi la coopération in vivo entre ces deux altérations génétiques. Des investigations supplémentaires des mécanismes moléculaires sous-jacents ont montré que le traitement d'astrocytes primaires (INK4a/Arf-KO, TP53-K0 et INK4a/Arf TP53-K0) avec un inhibiteur spécifique (tyrphostine AU 1478) de ΔEGFR(2-7) avaient un effet négatif sur la croissance cellulaire. Cependant, une réduction de la phosphorylation de p42/44-MAPK n'a été observée qu'en présence du gène INK4a/Arf intact. Cette résistance à la répression de l'activation des p42/44-MAPK a été confirmée dans les lignées de cellules tumorales dérivées des modèles animaux mis au point: INK4a/Arf-K0 exprimant ΔEGFR(2-7) et TP53-KO, respectivement. La perte du gène INK4a/Arf apparaît comme étant responsable de l'absence de modulation des signaux transmis par la voie d'EGFR. INK4a et/ou Arf pourrait donc jouer un rôle dans un mécanisme de feedback, contribuant ainsi à l'effet coopératif sélectionné dans la tumorigenèse des glioblastomes humains. La réponse aux traitements ciblés contre EGFR chez les patients souffrant de glioblastome pourrait donc ne pas dépendre de la seule expression d'EGFR/ΔEGFR(2-7), mais aussi de l'absence du gène INK4a/Arf. Ceci entraînant de sérieuses conséquences cliniques dans le développement de stratégies pour le traitement des glioblastomes humains.
Abstract
Glioblastoma multiforme is the most frequent and most the most malignant neoplasm of the human nervous system. The prognosis of patients with glioblastoma remains poor with a median survival of one year, even after surgery, chemotherapy and radiotherapy. Primary glioblastoma are characterized by frequent amplification of the epidermal growth factor receptor (egfr) gene or expression of its truncated mutant (ΔEGFR(2-7)) in association with the loss of the INK4a/Arf gene locus. Thus, suggesting a cooperative effect between an activated EGFR-pathway and the abrogation of INK4a/Arf.
Modeling this pathway in mice using a neurografting paradigm yielded glioma, at a low penetrance, expressing ΔEGFR(2-7) and carrying an inactive INK4a/Arf gene locus, hence, supporting the notion of a cooperative effect. Subsequent investigation of underlying molecular mechanisms using primary cortical astrocytes revealed that treatment with the specific EGFR inhibitor tyrphostin AG 1478 abolished growth induction mediated by ectopic expression of ΔEGFR(2-7) in INK4a/Arf-KO, TP53-KO, and INK4a/Arf-KO_TP53-K0 astrocytes. However, phosphorylation of the p42/44-MAPK was only reduced in the presence of an intact INK4a/Arf gene locus. This resistance to downregulate the MAPK-pathway in absence of INK4a/Arf was confirmed in cell lines derived from the mouse glioma with the respective initial genetic alterations, INK4a/Arf-K0 with ectopic expression of ΔEGFR(2-7), or TP53-KO, respectively. Thus, deletion of INK4a/Arf appears to modulate downstream signaling response in the EGFR—pathway, suggesting a new role for INK4a and /or Arf in a negative feedback loop that may contribute to the observed cooperative tumorigenic effect selected for in human glioblastoma. Clinical consequences may be that the response to treatments specifically targeting the EGFR in glioblastoma patients may not only depend on the egfr-status, but also on the INK4a/Arf-status.
Goal of this work
Glioblastoma multiforme (GBM) is the most malignant brain tumor and is associated with poor prognosis. A good understanding of genetic alterations and their association that lead to tumorigenesis is an essential prerequisite for the development of targeted therapies in the future.
We aimed to investigate the potential cooperative effect between INK4a/Arf deletion and egfr amplification/overexpression and their underlying molecular mechanisms in primary glioblastoma development. To achieve this goal, we followed three different approaches, generation of a glioblastoma mouse model, investigation of resistance to anoikis conferred by EGFR overexpression and INK4a/Arf loss, and inhibition of ΔEGFR(2-7) by small molecule drugs to study downstream pathways.
To establish a glioblastoma mouse model, we followed a transplantation paradigm, because mice constitutively lacking INK4a/Arf gene have a reduced life span due to the development of extra-cerebral lethal tumors, mainly lymphomas (1). To avoid these peripheral effects we have established an orthotopic transplantation model using neuroectodermal cells (embryonic day 13.5) hemizygous or deficient for INK4a/Arf, and transduced with EGFR or ΔEGFR(2-7). Based on the highly invasive character of glioblastoma, we studied the effect of INK4a/Arf loss associated to ΔEGFR(2-7) expression in resistance to anoikis. For this purpose, we used primary cortical astrocytes derived from newborn mice with the genotypes INK4a/Arf-K0 or TP53-KO, respectively, retroviraly transduced with EGFR or ΔEGFR(2-7). These cells were cultured in conditions that prevent cell adhesion and analyzed for cell survival and induction of apoptosis.
And finally, because of the relevance of EGFR inhibition in clinical trials, we investigated the influence of ΔEGFR(2-7) inhibition by tyrphostin AG 1478 on subsequent signaling pathways in cell lines derived from the glioma mouse model and primary INK4a/Arf-K0 or TP53-K0 astrocytes.
Create date
19/11/2007 13:39
Last modification date
29/05/2020 12:21
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