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The influence of AKT isoforms on radiation sensitivity and DNA repair in colon cancer cell lines
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. (Biomedicinsk strålningsvetenskap)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
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2014 (English)In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 35, no 4, 3525-3534 p.Article in journal (Refereed) Published
Abstract [en]

In response to ionizing radiation, several signaling cascades in the cell are activated to repair the DNA breaks, prevent apoptosis, and keep the cells proliferating. AKT is important for survival and proliferation and may also be an activating factor for DNA-PKcs and MRE11, which are essential proteins in the DNA repair process. AKT (PKB) is hyperactivated in several cancers and is associated with resistance to radiotherapy and chemotherapy. There are three AKT isoforms (AKT1, AKT2, and AKT3) with different expression patterns and functions in several cancer tumors. The role of AKT isoforms has been investigated in relation to radiation response and their effects on DNA repair proteins (DNA-PKcs and MRE11) in colon cancer cell lines. The knockout of AKT1 and/or AKT2 affected the radiation sensitivity, and a deficiency of both isoforms impaired the rejoining of radiation-induced DNA double strand breaks. Importantly, the active/phosphorylated forms of AKT and DNA-PKcs associate and exposure to ionizing radiation causes an increase in this interaction. Moreover, an increased expression of both DNA-PKcs and MRE11 was observed when AKT expression was ablated, yet only DNA-PKcs expression influenced AKT phosphorylation. Taken together, these results demonstrate a role for both AKT1 and AKT2 in radiotherapy response in colon cancer cells involving DNA repair capacity through the nonhomologous end joining pathway, thus suggesting that AKT in combination with DNA-PKcs inhibition may be used for radiotherapy sensitizing strategies in colon cancer.

Place, publisher, year, edition, pages
2014. Vol. 35, no 4, 3525-3534 p.
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:uu:diva-221446DOI: 10.1007/s13277-013-1465-9ISI: 000334495900084OAI: oai:DiVA.org:uu-221446DiVA: diva2:709104
Available from: 2014-03-31 Created: 2014-03-31 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Colorectal cancer and radiation response: The role of EGFR, AKT and cancer stem cell markers
Open this publication in new window or tab >>Colorectal cancer and radiation response: The role of EGFR, AKT and cancer stem cell markers
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The primary treatment for colorectal cancer is surgery. Radiotherapy and chemotherapy, sometimes combined, are also frequently used to diminish recurrence risk. In response to radiation exposure, several cellular signaling cascades are activated to repair DNA breaks, prevent apoptosis and to keep the cells proliferating. Several proteins in the radiation response and cell survival pathways are potential targets to enhance the effects of radiation. The epidermal growth factor receptor (EGFR), which is frequently upregulated in colorectal cancer and exhibits a radiation protective function, is an attractive target for treatment. EGFR is activated by radiation which in turn activates numerous signaling pathways such as the PI3 kinase/AKT cascade, the RAS/RAF/ERK pathway and STAT leading to tumor cell proliferation. EGFR is also believed to interact with proteins in the DNA repair process, such as DNA-PKcs and MRE11. The cytotoxic effect of an affibody molecule (ZEGFR:1907)2, with high affinity to EGFR,  in combination with radiation produced a small, but significant, reduction in survival in a KRAS mutated cell line. However, not in the BRAF mutated cell line. The next step was therefore to target proteins downstream of EGFR such as AKT. There was an interaction between AKT and the DNA repair proteins DNA-PKcs and MRE11 and both AKT1 and AKT2 were involved in the radiation response. The knockout of both AKT isoforms impaired the DNA double strand break rejoining after radiation and suppression of DNA-PKcs increased the radiations sensitivity and decreased the DNA repair further. The AKT isoforms also affected the expression of cancer stem cell markers CD133 and CD44 which are associated with the formation of metastasis as well as radiation and drug resistance. The CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. AKT was also involved in cell migration, cell-adhesion and metabolism. Overall, these results illustrate the complexity in response to radiation and drugs in cells with different mutations and the need for combining inhibitors against several targets such as EGFR, AKT, DNA-PKcs, CD133 or CD44. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 94 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 999
Keyword
colorectal cancer, radiation, AKT, EGFR, cancer stem cells, CD133, CD44
National Category
Cell Biology Biochemistry and Molecular Biology
Research subject
Biomedical Radiation Science; Biology with specialization in Molecular Cell Biology
Identifiers
urn:nbn:se:uu:diva-222836 (URN)978-91-554-8951-9 (ISBN)
Public defence
2014-06-05, Rudbecksalen, Dag Hammarskjöldsväg 20A, Uppsala, 14:00 (English)
Opponent
Supervisors
Available from: 2014-05-15 Created: 2014-04-14 Last updated: 2014-08-15Bibliographically approved
2. Radiation response in human cells: DNA damage formation, repair and signaling
Open this publication in new window or tab >>Radiation response in human cells: DNA damage formation, repair and signaling
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ionizing radiation induces a range of different DNA lesions. In terms of mutation frequency and mammalian cell survival, the most critical of these lesions is the DNA double-strand break (DSB). DSB left unrepaired or mis-repaired may result in chromosomal aberrations that can lead to permanent genetic changes or cell death. The complexity of the DNA damage and the capacity to repair the DSB will determine the fate of the cell. This thesis focuses on the DNA damage formation, repair and signaling after irradiation of human cells.

Radiation with high linear energy transfer (LET) produces clustered damaged sites in the DNA that are difficult for the cell to repair. Within these clustered sites, non-DSB lesions are formed that can be converted into a DSB and add to the damage complexity and affect DSB repair and the measurement. Heat-labile sites in DNA are converted into DSB at elevated temperatures. We show that heat-released DSB are formed post-irradiation with high-LET ions and increase the initial yield of DSB by 30%-40%, which is similar to yields induced by low-LET radiation.

DNA-PKcs, a central player in non-homologous end-joining (NHEJ), the major mammalian DSB repair pathway, has been found to be both up- and downregulated in different tumor types. In Paper II we show that low levels of DNA-PKcs lead to extreme radiosensitivity but, surprisingly, had no effect on the DSB repair. However, the fraction of cells in G2/M phase increased two-fold in cells with low levels of DNA-PKcs. The study continued in Paper IV, where cells were synchronized to unmask potential roles of DNA-PKcs in specific cell cycle phases. Irradiation of DNA-PKcs suppressed cells in the G1/S phase caused a delay in cell cycle progression and an increase in accumulation of G2 cells. Further, these cells showed defects in DNA repair, where a significant amount of 53BP1 foci remained after 72 h. This further strengthens the hypothesis that DNA-PKcs has a role in regulation of mitotic progression.

Several cellular signaling pathways are initiated in response to radiation. One of these downstream signaling proteins is AKT. We identified an interaction between DNA-PKcs and AKT. Knockouts of both AKT1 and AKT2 impaired DSB rejoining after radiation and low levels of DNA-PKcs increased radiosensitivity and decreased DNA repair further.  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 52 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1157
Keyword
DNA damage, DNA repair, DSB, NHEJ, DNA-PK, ionizing radiation, heat-labile sites
National Category
Other Basic Medicine Cell and Molecular Biology Cell Biology
Research subject
Biomedical Radiation Science
Identifiers
urn:nbn:se:uu:diva-265137 (URN)978-91-554-9397-4 (ISBN)
Public defence
2015-12-16, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds v 20, Uppsala, 14:00 (English)
Opponent
Supervisors
Available from: 2015-11-24 Created: 2015-10-22 Last updated: 2016-01-13

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Häggblad Sahlberg, SaraGustafsson, Ann-SofieGlimelius, BengtStenerlöw, Bo

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