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FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma
Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Karolinska Inst, Dept Cell & Mol Biol, Stockholm, Sweden.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.ORCID iD: 0000-0003-2835-1518
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2016 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 35, no 20, 2192-2212 p.Article in journal (Refereed) Published
Abstract [en]

SOX9 is a master transcription factor that regulates development and stem cell programs. However, its potential oncogenic activity and regulatory mechanisms that control SOX9 protein stability are poorly understood. Here, we show that SOX9 is a substrate of FBW7, a tumor suppressor, and a SCF (SKP1/CUL1/F-box)-type ubiquitin ligase. FBW7 recognizes a conserved degron surrounding threonine 236 (T236) in SOX9 that is phosphorylated by GSK3 kinase and consequently degraded by SCFFBW7 alpha. Failure to degrade SOX9 promotes migration, metastasis, and treatment resistance in medulloblastoma, one of the most common childhood brain tumors. FBW7 is either mutated or downregulated in medulloblastoma, and in cases where FBW7 mRNA levels are low, SOX9 protein is significantly elevated and this phenotype is associated with metastasis at diagnosis and poor patient outcome. Transcriptional profiling of medulloblastoma cells expressing a degradation-resistant SOX9 mutant reveals activation of pro-metastatic genes and genes linked to cisplatin resistance. Finally, we show that pharmacological inhibition of PI3K/AKT/mTOR pathway activity destabilizes SOX9 in a GSK3/FBW7-dependent manner, rendering medulloblastoma cells sensitive to cytostatic treatment.

Place, publisher, year, edition, pages
2016. Vol. 35, no 20, 2192-2212 p.
Keyword [en]
FBW7, SOX9, Medulloblastoma, FBXW7, ubiquitin, migration, metastasis, drug resistance
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-274626DOI: 10.15252/embj.201693889ISI: 000385708000004OAI: oai:DiVA.org:uu-274626DiVA: diva2:897102
Funder
Swedish Childhood Cancer FoundationSwedish Cancer SocietySwedish Research CouncilEU, European Research Council, 640275Ragnar Söderbergs stiftelseSwedish Society of MedicineÅke Wiberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceThe Karolinska Institutet's Research Foundation
Note

Aldwin Suryo Rahmanto and Vasil Savov contributed equally to this work as first authors

Andrä Brunner, Sara Bolin and Holger Weishaupt contributed equally to this work as second authors

Fredrik J Swartling and Olle Sangfelt contributed equally to this work as corresponding authors

Available from: 2016-01-24 Created: 2016-01-24 Last updated: 2016-11-21Bibliographically approved
In thesis
1. The Role of SOX9 in Medulloblastoma
Open this publication in new window or tab >>The Role of SOX9 in Medulloblastoma
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Overall survival is about 70% and in cases where current treatment fails, the disease recurs and most often is fatal. At the molecular level, MB can be divided into four defined subgroups: WNT, SHH, Group 3 and Group 4. Amplification of MYC family genes is common in MB and correlates with poor prognosis and tumor relapse.

In this thesis we showed how MYCN initiates brain tumors when transduced in neural stem cells (NSCs). Prior to transduction, NSCs were isolated from different brain regions and at various time points. While overexpression of wild-type MYCN did not generate any tumors, orthotopic transplantation of MYCNT58A-expressing forebrain, brain stem and cerebellar NSCs induced diffuse malignant glioma, PNET-like tumors and MB, respectively. Interestingly, MYCNT58A-expressing cerebellar NSCs induced SHH-dependent MB from embryonic cells but SHH-independent MB from postnatal cells. We further showed that cerebellar NSCs transduced with both MYCNT58A and transcription factor SOX9 developed tumors faster and promoted distant migration into the forebrain.

The function and regulation of SOX9 in MB cells is poorly understood. We identified SOX9 protein as target of FBW7 ubiquitin ligase and demonstrated the effects of SOX9 on MB cells migration, metastasis and drug resistance. We further blocked PI3K pathway to destabilize SOX9 which sensitized cells to cytostatic treatment.

We used a (TetOFF) transgenic mouse model of MYCN-induced MB (GTML) and crossed it with a (TetON) transgene which allowed us to specifically target rare SOX9-positive cells in the tumor. In this system, MB develops spontaneously and SOX9-negative tumor cells can be killed off by doxycycline. The few remaining SOX9-positive cancer cells were able to promote distant MB recurrences. Such a pattern of relapse was recently shown for Group 3 and 4 human MB where about 90% of the recurrences were distant.

In summary, this thesis demonstrates that MYCN can generate various types of brain tumors depending on the timing and location of its expression. It further defines the existence of a rare population of SOX9-expressing MB cells that are involved in causing distant MB recurrences. Finally, it describes how SOX9 is stabilized in MB cells and increases MB migration and therapy resistance.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 39 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1175
Keyword
Medulloblastoma, SOX9, MYCN, cancer development, recurrence, regulation, tumor metastasis, migration
National Category
Cell and Molecular Biology Cancer and Oncology Pediatrics
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-274630 (URN)978-91-554-9461-2 (ISBN)
Public defence
2016-03-11, Rudbecksalen, Dag Hammarskjоlds vаg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2016-02-18 Created: 2016-01-24 Last updated: 2016-03-09
2. Mechanisms of Medulloblastoma Dissemination and Novel Targeted Therapies
Open this publication in new window or tab >>Mechanisms of Medulloblastoma Dissemination and Novel Targeted Therapies
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Medulloblastomas are the most frequent malignant childhood brain tumors, arising in the posterior fossa of children. The overall 5-year survival is 70%, although children often suffer severe long-term side effects from standard medical care. To improve progression-free survival and quality of life for these children, finding new therapeutic targets in medulloblastoma is imperative.

Medulloblastoma is divided in to four molecular subgroups (WNT, SHH, Group 3 and Group 4) based on key developmental pathways essential for the initiation and maintenance of tumor development. The MYC family of proto-oncogenes regulates cell proliferation and differentiation in normal brain. Aberrant expression of MYC proteins occurs commonly in medulloblastoma.

Our studies on Group 3 medulloblastoma identify the transcription factor SOX9 as a novel target for the E3 ubiquitin ligase FBW7, and show that increased stability of SOX9 confers an increased metastatic potential in medulloblastoma. Moreover, SOX9-positive cells drive distant recurrences in medulloblastoma when combining two regulatable TetON/OFF systems. MYCN depletion leads to increased SOX9 expression in Group 3 medulloblastoma cells, and the recurring tumor cells are more migratory in vitro and in vivo. Segueing to treatment of medulloblastoma, we show that BET bromodomain inhibition specifically targets MYC-amplified medulloblastoma cells by downregulating MYC and MYC-transcriptional targets, and that combining BET bromodomain- and cyclin-dependent kinase- inhibition improves survival in mice compared to single therapy. Combination treatment results in decreased MYC levels and increased apoptosis, and RNA-seq confirms upregulation of apoptotic markers along with downregulated MYC target genes in medulloblastoma cells.

This thesis addresses novel findings in transcription factor biology, recurrence and treatment in Group 3 medulloblastoma, the most malignant subgroup of the disease.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 49 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1254
Keyword
Medulloblastoma, Recurrence, MYC, SOX9, FBW7, Treatment, BET bromodomains, Cyclin-dependent kinases
National Category
Basic Medicine
Research subject
Oncology
Identifiers
urn:nbn:se:uu:diva-300907 (URN)978-91-554-9692-0 (ISBN)
Public defence
2016-11-04, Rudbecksalen, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2016-10-12 Created: 2016-08-15 Last updated: 2016-10-19

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Savov, VasilBolin, SaraWeishaupt, HolgerRosén, GabrielaČančer, MatkoHutter, SonjaSwartling, Fredrik J.
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