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Colorectal cancer and radiation response: The role of EGFR, AKT and cancer stem cell markers
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science. (Biomedicinsk strålningsvetenskap)
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 [en]
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: urn:nbn:se:uu:diva-222836ISBN: 978-91-554-8951-9 (print)OAI: oai:DiVA.org:uu-222836DiVA: diva2:712377
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
List of papers
1. The effect of a dimeric Affibody molecule (ZEGFR:1907)2 targeting EGFR in combination with radiation in colon cancer cell lines
Open this publication in new window or tab >>The effect of a dimeric Affibody molecule (ZEGFR:1907)2 targeting EGFR in combination with radiation in colon cancer cell lines
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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.

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:nbn:se:uu:diva-165752 (URN)10.3892/ijo.2011.1177 (DOI)000297403800022 ()21879255 (PubMedID)
Note

Online ISSN:1791-2423

Available from: 2012-01-09 Created: 2012-01-09 Last updated: 2017-12-08Bibliographically approved
2. The influence of AKT isoforms on radiation sensitivity and DNA repair in colon cancer cell lines
Open this publication in new window or tab >>The influence of AKT isoforms on radiation sensitivity and DNA repair in colon cancer cell lines
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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.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-221446 (URN)10.1007/s13277-013-1465-9 (DOI)000334495900084 ()
Available from: 2014-03-31 Created: 2014-03-31 Last updated: 2017-12-05Bibliographically approved
3. Evaluation of cancer stem cell markers CD133, CD44, CD24: association with AKT isoforms and radiation resistance in colon cancer cells.
Open this publication in new window or tab >>Evaluation of cancer stem cell markers CD133, CD44, CD24: association with AKT isoforms and radiation resistance in colon cancer cells.
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 4, e94621- p.Article in journal (Refereed) Published
Abstract [en]

The cell surface proteins CD133, CD24 and CD44 are putative markers for cancer stem cell populations in colon cancer, associated with aggressive cancer types and poor prognosis. It is important to understand how these markers may predict treatment outcomes, determined by factors such as radioresistance. The scope of this study was to assess the connection between EGFR, CD133, CD24, and CD44 (including isoforms) expression levels and radiation sensitivity, and furthermore analyze the influence of AKT isoforms on the expression patterns of these markers, to better understand the underlying molecular mechanisms in the cell. Three colon cancer cell-lines were used, HT-29, DLD-1, and HCT116, together with DLD-1 isogenic AKT knock-out cell-lines. All three cell-lines (HT-29, HCT116 and DLD-1) expressed varying amounts of CD133, CD24 and CD44 and the top ten percent of CD133 and CD44 expressing cells (CD133(high)/CD44(high)) were more resistant to gamma radiation than the ten percent with lowest expression (CD133(low)/CD44(low)). The AKT expression was lower in the fraction of cells with low CD133/CD44. Depletion of AKT1 or AKT2 using knock out cells showed for the first time that CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. There were several genes in the cell adhesion pathway which had significantly higher expression in the AKT2 KO cell-line compared to the AKT1 KO cell-line; however important genes in the epithelial to mesenchymal transition pathway (CDH1, VIM, TWIST1, SNAI1, SNAI2, ZEB1, ZEB2, FN1, FOXC2 and CDH2) did not differ. Our results demonstrate that CD133(high)/CD44(high) expressing colon cancer cells are associated with AKT and increased radiation resistance, and that different AKT isoforms have varying effects on the expression of cancer stem cell markers, which is an important consideration when targeting AKT in a clinical setting.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-221449 (URN)10.1371/journal.pone.0094621 (DOI)000335298200022 ()
Available from: 2014-03-31 Created: 2014-03-31 Last updated: 2017-12-05Bibliographically approved
4. Different functions of AKT1 and AKT2 in molecular pathways, cell migration and metabolism in colon cancer cells
Open this publication in new window or tab >>Different functions of AKT1 and AKT2 in molecular pathways, cell migration and metabolism in colon cancer cells
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2017 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 50, no 1, 5-14 p.Article in journal (Refereed) Published
Abstract [en]

AKT is a central protein in many cellular pathways such as cell survival, proliferation, glucose uptake, metabolism, angiogenesis, as well as radiation and drug response. The three isoforms of AKT (AKT1, AKT2 and AKT3) are proposed to have different physiological functions, properties and expression patterns in a cell type-dependent manner. As of yet, not much is known about the influence of the different AKT isoforms in the genome and their effects in the metabolism of colorectal cancer cells. In the present study, DLD-1 isogenic AKT1, AKT2 and AKT'/2 knockout colon cancer cell lines were used as a model system in conjunction with the parental cell line in order to further elucidate the differences between the AKT isoforms and how they are involved in various cellular pathways. This was done using genome wide expression analyses, metabolic profiling and cell migration assays. In conclusion, downregulation of genes in the cell adhesion, extracellular matrix and Notch-pathways and upregulation of apoptosis and metastasis inhibitory genes in the p53-pathway, confirm that the knockout of both AKT1 and AKT2 will attenuate metastasis and tumor cell growth. This was verified with a reduction in migration rate in the AKT1 KO and AKT2 KO and most explicitly in the AKT1/2 KO. Furthermore, the knockout of AKT1, AKT2 or both, resulted in a reduction in lactate and alanine, suggesting that the metabolism of carbohydrates and glutathione was impaired. This was further verified in gene expression analyses, showing downregulation of genes involved in glucose metabolism. Additionally, both AKT1 KO and AKT2 KO demonstrated an impaired fatty acid metabolism. However, genes were upregulated in the Wnt and cell proliferation pathways, which could oppose this effect. AKT inhibition should therefore be combined with other effectors to attain the best effect.

Keyword
Microarray, metabolism, cell migration AKT1, AKT2, AKT, PKB, gene expression, colon-cancer, DLD-1, metabolomics, CD44, CD133
National Category
Biochemistry and Molecular Biology
Research subject
Biomedical Radiation Science; Biology with specialization in Molecular Cell Biology; Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-222834 (URN)10.3892/ijo.2016.3771 (DOI)000391419200001 ()
Available from: 2014-04-14 Created: 2014-04-14 Last updated: 2017-12-05Bibliographically approved

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Häggblad Sahlberg, Sara

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