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The effect of a dimeric Affibody molecule (ZEGFR:1907)2 targeting EGFR in combination with radiation 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. (BMS)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. (BMS)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
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2012 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 40, no 1, 176-184 p.Article in journal (Refereed) Published
Abstract [en]

The epidermal growth factor receptor (EGFR) is frequently overexpressed in colorectal cancer and is therefore an attractive target for treatment. (ZEGFR:1907)2 is a newly developed dimeric affibody molecule with high affinity to the extracellular part of EGFR. In this study, we evaluated the cytotoxic effects of (ZEGFR:1907)2 in combination with external radiation and the possible inhibitory effects in the EGFR signalling pathways in the colon cancer cell lines HT-29 and HCT116. The effects were compared with an EGFR antibody (cetuximab) and the tyrosine kinase inhibitors (erlotinib and sunitinib). These cell lines are genotypically different with respect to e.g. KRAS and BRAF mutational status, recently shown to be of clinical significance for therapeutic effects. Both cell lines express approximately 100,000-150,000 EGFRs per cell but differ in the radiation response (HCT116, SF2=0.28 and HT-29, SF2=0.70). Exposure to (ZEGFR:1907)2 produced a small, but significant, reduction in survival in HCT116 but did not affect HT-29 cells. Similar results were obtained after exposure to EGF and the EGFR antibody cetuximab. The EGFR tyrosine kinase targeting inhibitor erlotinib and the multi-tyrosine kinase inhibitor sunitinib reduced survival in both cell lines. However, none of the drugs had any significant radiosensitizing effects in combination with radiation. Akt and Erk are central proteins in the EGFR downstream signalling and in the cellular response to ionizing radiation. The activation of Akt (Ser 473) and Erk (Thr202/Tyr204) by radiation was both dose- and time-dependent. However the activation of EGFR was not clearly affected by radiation. Neither (ZEGFR:1907)2 nor any of the other drugs were able to completely inactivate Akt or Erk. On the contrary, erlotinib stimulated Akt phosphorylation in both cell lines and in HCT116 cells Erk was activated. Overall the results illustrate the complexity in response to radiation and drugs in cells with differential phenotypic status.

Place, publisher, year, edition, pages
2012. Vol. 40, no 1, 176-184 p.
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biology with specialization in Molecular Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-165752DOI: 10.3892/ijo.2011.1177ISI: 000297403800022PubMedID: 21879255OAI: oai:DiVA.org:uu-165752DiVA: diva2:474600
Note

Online ISSN:1791-2423

Available from: 2012-01-09 Created: 2012-01-09 Last updated: 2017-12-08Bibliographically 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)
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Available from: 2014-05-15 Created: 2014-04-14 Last updated: 2014-08-15Bibliographically approved

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Häggblad Sahlberg, SaraSpiegelberg, DianaLennartsson, JohanNygren, PeterGlimelius, BengtStenerlöw, Bo

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