molecular mechanisms of imatinib resistance in gastrointestinal stromal tumor with focus on ...

92 Pages·2014·2.22 MB·English

From DEPARTMENT OF ONCOLOGY-PATHOLOGY
Karolinska Institutet, Stockholm, Sweden
MOLECULAR MECHANISMS OF
IMATINIB RESISTANCE IN
GASTROINTESTINAL STROMAL TUMOR
WITH FOCUS ON MICRORNAS

Pınar Akçakaya

Stockholm 2015
All previously published papers were reproduced with permission from the publisher.
Published by Karolinska Institutet.
Cover artwork by Emre Özdamarlar, Resistance against the “magic bullet”.
Printed by Eprint AB, Oxtorgsgatan 9-11, 111 57 Stockholm
© Pınar Akçakaya, 2015
ISBN 978-91-7549-730-3
Molecular mechanisms of imatinib resistance in
gastrointestinal stromal tumor with focus on
microRNAs

Thesis for doctoral degree (Ph.D.)
Radiumhemmets föreläsningssal, P1:01, Karolinska University Hospital Solna,
Stockholm
Friday January 9th 2015, at 09:30
By
Pınar Akçakaya
Principal Supervisor: Opponent:
Weng-Onn Lui, Associate Professor Özgür Şahin, Assistant Professor
Karolinska Institutet Bilkent University
Department of Oncology-Pathology Department of Molecular Biology and Genetics

Co-supervisors: Examination Board:
Catharina Larsson, Professor Otte Brosjö, Associate Professor
Karolinska Institutet Karolinska Institutet
Department of Oncology-Pathology Department of Molecular Medicine and Surgery

Robert Bränström, Associate Professor Xiao-Feng Sun, Professor
Karolinska Institutet Linköping University
Department of Molecular Medicine and Surgery Department of Clinical and Experimental
Medicine
Arne Östman, Professor
Karolinska Institutet Leif Stenke, Associate Professor
Department of Oncology-Pathology Karolinska Institutet
Departments of Medicine and Oncology-Pathology

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ABSTRACT
Gastrointestinal stromal tumor (GIST) is mainly initialized by mutations in receptor tyrosine
kinase genes KIT or PDGFRA. The development of imatinib, a small molecule inhibitor that
targets these tyrosine kinase receptors, remarkably improved patient outcome. However,
imatinib resistance remains a major therapeutic challenge in GIST therapy, and its underlying
mechanisms are still not completely understood. This thesis work aimed to explore the role of
microRNAs (miRNAs) and DOG1 in imatinib resistance of GIST.
In Paper I, we identified specific miRNA signatures associated with imatinib resistance,
metastatic disease, KIT mutational status and survival in GIST patients treated with
neoadjuvant imatinib. Importantly, we demonstrate that miR-125a-5p modulates imatinib
response in the single KIT-mutated GIST882 cells through PTPN18 regulation. This study
highlights the clinical impact of miRNAs in GIST patients treated with imatinib pre-
operatively, and suggests the important role of miR-125a-5p and PTPN18 in imatinib
resistance of GISTs.
In Paper II, we tested our hypothesis that miR-125a-5p overexpression in imatinib-resistant
GISTs suppresses PTPN18 expression that subsequently leads to defective FAK
dephosphorylation. Indeed, we demonstrate that silencing of PTPN18 increased FAK
phosphorylation in GIST cells, and the acquired imatinib-resistant GIST882R cells exhibited
higher pFAK and lower PTPN18 expressions than the imatinib-sensitive parental cells. FAK
and pFAK expressions are also associated with imatinib resistance in GIST specimens. This
study highlights the potential role of PTPN18 and pFAK in imatinib resistance of GIST.
In Paper III, we found that miR-320a and miR-320b are upregulated and their potential
target MCL1 is downregulated in imatinib-treated GISTs. Imatinib treatment affects MCL1
and miR-320 levels in GIST882 cells, and the imatinib-resistant GIST882R cells showed
higher levels of the anti-apoptotic MCL1L isoform and lower expression of miR-320a/b as
compared to GIST882 cells. This study suggests that miR-320a/b and MCL1 play a role in
imatinib-induced cell death and resistance in GIST.
In Paper IV, we evaluated the functional role of DOG1 in imatinib-resistant GIST48 and –
sensitive GIST882 cells using specific DOG1 activator and inhibitor. We showed that DOG1
is localized in different cellular compartments in imatinib-resistant and -sensitive GIST
cells. Pharmacological modulation of DOG1 activity has subtle effect on cell viability and
proliferation, but may shift early apoptotic cells to late apoptotic stages in GIST48 cells.
Overall, this thesis work describes the role of miRNAs in cell viability and resistance to
imatinib treatment in GIST.

LIST OF PUBLICATIONS

The thesis is based on the following papers, which will be referred by their Roman numerals
throughout the text.

I. Akçakaya P*, Caramuta S, Åhlén J, Ghaderi M, Berglund E, Östman A, Bränström R,
Larsson C and Lui WO*.
microRNA expression signatures of gastrointestinal stromal tumors: associations to
imatinib resistance and patient outcome.
British Journal of Cancer 2014; 111(11):2091-2102

II. Akçakaya P*, Gangaev A, Lee L, Zeljic K, Hajeri P, Berglund E, Ghaderi M, Åhlén
J, Bränström R, Östman A, Larsson C and Lui WO*.
FAK phosphorylation is regulated by PTPN18 and associated with imatinib resistance in
gastrointestinal stromal tumors.
Manuscript

III. Akçakaya P*, Zeljic K, Åhlén J, Gangaev A, Ghaderi M, Caramuta S, Berglund E,
Bränström R, Larsson C and Lui WO*.
Involvement of MCL1 and miR-320 in imatinib-induced cell death of gastrointestinal
stromal tumor cells.
Manuscript

IV. Berglund E*, Akçakaya P, Berglund D, Karlsson F, Vukojević V, Lee L, Bogdanović
D, Lui WO, Larsson C, Zedenius J, Fröbom R, Bränström R.
Functional role of the Ca2+-activated Cl- channel DOG1/TMEM16A in gastrointestinal
stromal tumor cells.
Experimental Cell Research 2014; 326(2):315-325

* Corresponding author
LIST OF RELATED PUBLICATIONS

1. Berglund E, Ubhayasekera SJ, Karlsson F, Akçakaya P, Aluthgedara W, Åhlén J, Fröbom
R, Nilsson IL, Lui WO, Larsson C, Zedenius J, Bergquist J, Bränström R.
Intracellular concentration of the tyrosine kinase inhibitor imatinib in gastrointestinal stromal
tumor cells.
Anticancer Drugs 2014; 25(4):415-422.

2. Caramuta S, Lee L, Özata DM, Akçakaya P, Georgii-Hemming P, Xie H, Amini RM,
Lawrie CH, Enblad G, Larsson C, Berglund M, Lui WO.
Role of microRNAs and microRNA machinery in the pathogenesis of diffuse large B-cell
lymphomas.
Blood Cancer Journal 2013; 3:e152.

3. Caramuta S, Lee L, Özata DM, Akçakaya P, Xie H, Höög A, Zedenius J, Bäckdahl M,
Larsson C, Lui WO.
Clinical and functional impact of TRBP2 over-expression in adrenocortical carcinoma.
Endocrine Related Cancer 2013; 20(4): 551-564.

4. Berglund E, Berglund D, Akçakaya P, Ghaderi M, Daré E, Berggren PO, Köhler M,
Aspinwall CA, Lui WO, Zedenius J, Larsson C, Bränström R.
Evidence for Ca(2+)-regulated ATP release in gastrointestinal stromal tumors.
Experimental Cell Research 2013; 319(8):1229-1238.

5. Özata DM, Caramuta S, Velázquez-Fernández D, Akçakaya P, Xie H, Höög A, Zedenius J,
Bäckdahl M, Larsson C, Lui WO.
The role of microRNA deregulation in the pathogenesis of adrenocortical carcinoma.
Endocrine Related Cancer 2011; 18(6):643-655.

6. Akçakaya P, Ekelund S, Kolosenko I, Caramuta S, Özata DM, Xie H, Lindforss U,
Olivecrona H, Lui WO.
miR-185 and miR-133b deregulation is associated with overall survival and metastasis in
colorectal cancer.
International Journal of Oncology 2011; 39(2):311-318.