Krishnamoorthy, Gnana Prakasam
(2013)
HSP90 inhibition mediated AXL receptor tyrosine kinase downregulation-molecular mechanism and therapeutic implication.
[Tesi di dottorato]
| Tipologia del documento: |
Tesi di dottorato
|
| Lingua: |
English |
| Titolo: |
HSP90 inhibition mediated AXL receptor tyrosine kinase downregulation-molecular mechanism and therapeutic implication. |
| Autori: |
| Autore | Email |
|---|
| Krishnamoorthy, Gnana Prakasam | gnanaprakasam_23@yahoo.com |
|
| Data: |
1 Aprile 2013 |
| Numero di pagine: |
88 |
| Istituzione: |
Università degli Studi di Napoli Federico II |
| Dipartimento: |
Medicina Molecolare e Biotecnologie Mediche |
| Scuola di dottorato: |
Medicina molecolare |
| Dottorato: |
Oncologia ed endocrinologia molecolare |
| Ciclo di dottorato: |
25 |
| Coordinatore del Corso di dottorato: |
| nome | email |
|---|
| Santoro, Massimo | masantor@unina.it |
|
| Tutor: |
| nome | email |
|---|
| Carlomagno, Francesca | francesca.carlomagno@unina.it | | Melillo, Rosa Marina | rosmelil@unina.it |
|
| Data: |
1 Aprile 2013 |
| Numero di pagine: |
88 |
| Parole chiave: |
AXL; HSP90; 17-AAG. |
| Settori scientifico-disciplinari del MIUR: |
Area 06 - Scienze mediche > MED/04 - Patologia generale |
[error in script]
[error in script]
| Depositato il: |
11 Apr 2013 14:06 |
| Ultima modifica: |
17 Giu 2014 06:04 |
| URI: |
http://www.fedoa.unina.it/id/eprint/9327 |
Abstract
The Tyrosine kinase receptor AXL belongs to the TAM (Tyro3, AXL and Mer) family, whose members are characterized by a highly conserved kinase domain and adhesion molecule-like domains in the extracellular region. There are several reports showing AXL overexpression in cancer and its transforming potential, depicting its ability to drive tumor formation, progression and resistance to different anti-cancer agents; hence, AXL receptor is a clinically validated cancer target. Heat shock protein 90 (HSP90) is a critical player in regulating the maturation, stability, and activity of its client proteins. HSP90 inhibition causes depletion of multiple oncogenic client proteins, leading to blockade of many key cancer causing pathways. HSP90 inhibitors that derived from geldanamycin, such as 17-AAG, have emerged as promising therapeutic agents for cancer treatment and few of them have entered clinical trials.
In this study we show that AXL is a novel client of HSP90, as the two structurally unrelated HSP90 inhibitors Radicicol or 17-AAG, induced a time- and dose-dependent downregulation of endogenous or ectopically expressed AXL protein. Using biotin and 35S-methionine labelling of the biogenesis/trafficking receptor upon 17-AAG treatment we showed that 17-AAG induced depletion of AXL on the plasma membrane, by mediating degradation of fully glycosylated AXL receptor from internal organelles and therefore restricting its transport to the cell surface. We demonstrated that 17-AAG induced AXL polyubiquitination and subsequent degradation via proteasome. HSP90 functions in the context of a multi-protein chaperone complex. By co-immunoprecipitation experiments, interactions between AXL and the components of chaperone complex like HSP90 itself, HSP70, and Ubiquitin E3 ligase CHIP (Carboxy terminus of Hsc70 Interacting Protein) were shown to be modulated by 17-AAG treatment. By contrast to wt CHIP, the functionally inactive CHIP mutant, CHIP K30A failed to accumulate AXL polyubiquitinated species even in the presence of 17-AAG, suggesting the involvement of endogenous CHIP in 17-AAG induced AXL polyubiquitination. We could also show that, it is the intracellular domain (ICD) of AXL containing the TK domain that is crucial to acquire 17-AAG sensitivity, as the AXL ICD-deleted mutant is insensitive to 17-AAG mediated degradation. However, AXL kinase activity is not required for 17-AAG mediated AXL loss, since inhibition of kinase activity by Bosutinib did not affect AXL sensitivity to 17-AAG. Similarly, AXL kinase dead mutant was as sensitive as the wt receptor to 17-AAG induced degradation. Finally, we also demonstrated that 17-AAG induced AXL downregulation could interfere with AXL mediated biological activity, measured as activation of transcription from the AP1 promoter, suggesting a possible therapeutic approach to counteract AXL driven oncogenic activity or drug resistance. Our data convincingly clarify the molecular basis of AXL downregulation by HSP90 inhibitor 17-AAG and suggest that HSP90 inhibition in anti-cancer therapy can exert its effect through inhibition of multiple kinases, including AXL itself.
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