| 1. |
- Akçakaya, Pınar
(författare)
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Molecular mechanisms of imatinib resistance in gastrointestinal stromal tumor with focus on microRNAs
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.
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| 2. |
- Berglund, Erik, et al.
(författare)
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Evidence for Ca2+-regulated ATP release in gastrointestinal stromal tumors
- 2013
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 319:8, s. 1229-1238
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Tidskriftsartikel (refereegranskat)abstract
- Gastrointestinal stromal tumors (GISTs) are thought to originate from the electrically active pacemaker cells of the gastrointestinal tract. Despite the presence of synaptic-like vesicles and proteins involved in cell secretion it remains unclear whether GIST cells possess regulated release mechanisms. The GIST tumor cell line GIST882 was used as a model cell system, and stimulus-release coupling was investigated by confocal microscopy of cytoplasmic free Ca2+ concentration ([Ca(2+)1](i)), flow cytometry, and luminometric measurements of extracellular ATP. We demonstrate that GIST cells have an intact intracellular Ca2+-signaling pathway that regulates ATP release. Cell viability and cell membrane integrity was preserved, excluding ATP leakage due to cell death and suggesting active ATP release. The stimulus-secretion signal transduction is at least partly dependent on Ca2+ influx since exclusion of extracellular Ca2+ diminishes the ATP release. We conclude that measurements of ATP release in GISTs may be a useful tool for dissecting the signal transduction pathway, mapping exocytotic components, and possibly for the development and evaluation of drugs. Additionally, release of ATP from GISTs may have importance for tumor tissue homeostasis and immune surveillance escape.
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| 3. |
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| 4. |
- Berglund, Erik, et al.
(författare)
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Functional role of the Ca2+-activated Cl- channel DOG1/TMEM16A in gastrointestinal stromal tumor cells
- 2014
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 326:2, s. 315-325
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Tidskriftsartikel (refereegranskat)abstract
- DOG1, a Ca2+-activated Cl- channel (CaCC), was identified in 2004 to be robustly expressed in gastrointestinal stromal tumors (GIST). It was rapidly included as a tumor marker in routine diagnostics, but the functional role remained unknown. CaCCs are important regulators of normal physiological functions, but also implicated in tumorigenesis, cancer progression, metastasis, cell migration, apoptosis, proliferation and viability in several malignancies. We therefore investigated whether DOG1 plays a role in the three latter in GIST by utilizing in vitro cell model systems. Confocal microscopy identified different subcellular localizations of DOG1 in imatinib-sensitive and imatinib-resistant cells. Electrophysiological studies confirmed that DOG1-specific pharmacological agents possess potent activating and inhibiting properties. Proliferation assays showed small effects up to 72 h, and flow cytometric analysis of adherent cells with 7-AAD/Annexin V detected no pharmacological effects on viable GIST cells. However, inhibition of DOG1 conveyed pro-apoptotic effects among early apoptotic imatinib-resistant cells. In conclusion, DOG1 generates Cl- currents in GIST that can be regulated pharmacologically, with small effects on cell viability and proliferation in vitro. Inhibition of DOG1 might act pro-apoptotic on some early apoptotic GIST cell populations. Further studies are warranted to fully illuminate the function of DOG1 and its potential as therapeutic target.
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| 5. |
- Berglund, Erik, et al.
(författare)
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Intracellular concentration of the tyrosine kinase inhibitor imatinib in gastrointestinal stromal tumor cells.
- 2014
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Ingår i: Anti-Cancer Drugs. - 0959-4973 .- 1473-5741. ; 25:4, s. 415-22
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Tidskriftsartikel (refereegranskat)abstract
- Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm in the gastrointestinal tract. In most GISTs, the underlying mechanism is a gain-of-function mutation in the KIT or the PDGFRA gene. Imatinib is a tyrosine kinase inhibitor that specifically blocks the intracellular ATP-binding sites of these receptors. A correlation exists between plasma levels of imatinib and progression-free survival, but it is not known whether the plasma concentration correlates with the intracellular drug concentration. We determined intracellular imatinib levels in two GIST cell lines: the imatinib-sensitive GIST882 and the imatinib-resistant GIST48. After exposing the GIST cells to imatinib, the intracellular concentrations were evaluated using LC-MS (TOF). The concentration of imatinib in clinical samples from three patients was also determined to assess the validity and reliability of the method in the clinical setting. Determination of imatinib uptake fits within detection levels and values are highly reproducible. The GIST48 cells showed significantly lower imatinib uptake compared with GIST882 in therapeutic doses, indicating a possible difference in uptake mechanisms. Furthermore, imatinib accumulated in the tumor tissues and showed intratumoral regional differences. These data show, for the first time, a feasible and reproducible technique to measure intracellular imatinib levels in experimental and clinical settings. The difference in the intracellular imatinib concentration between the cell lines and clinical samples indicates that drug transporters may contribute toward resistance mechanisms in GIST cells. This highlights the importance of further clinical studies to quantify drug transporter expression and measure intracellular imatinib levels in GIST patients.
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| 6. |
- Bestas, Burcu, et al.
(författare)
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A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo
- 2023
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Ingår i: NATURE COMMUNICATIONS. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9's off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes. SpCas9 unintended editing is a major concern. Here the authors report an off-target method using Duplex Sequencing with increased sensitivity for Cas9 mutation detection; they also identify a Cas9 variant of the II-B subfamily with intrinsic high fidelity (PsCas9) and see improved specificity.
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