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Different functions of AKT1 and AKT2 in molecular pathways, cell migration and metabolism in colon cancer cells.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science. (Biomedicinsk strålningsvetenskap)
(English)Manuscript (preprint) (Other academic)
Abstract [en]

AKT is a central protein in many different cellular pathways, which involves cell survival, proliferation, glucose uptake, metabolism, angiogenesis, radiation response 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. But not much is known about the influence of the different AKT isoforms in the whole genome and their effect in metabolism in colorectal cancer cells. We have previously shown that both AKT1 and AKT2 interact with the DNA-repair protein DNA-PKcs and that disruption of AKT1 and AKT2 increased the radiation sensitivity as well as influenced the expression of cancer stem cell markers CD44 and CD133.

In this study, the DLD-1 isogenic AKT 1-, AKT2-, and AKT1/2 knockout colon cancer cell lines were used as a model system together 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 whole genome expression analyses, metabolomic analyses, and cell migration assays.

In conclusion, the downregulation of genes in the cell adhesion-, extracellular matrix- and Notch-pathway as well as upregulation of apoptosis and metastasis inhibitory genes in the p53-pathway confirms that the knockout of both AKT1 and 2 will attenuate metastasis and tumor cell growth. This was verified with a reduction in migration rate in the AKT1 KO and AKT2 KO, but mostly in the AKT1/2 KO. Furthermore, the knockout of AKT1, AKT2 or both, resulted in a reduction in lactate and alanine which suggests that the metabolism of carbohydrates and glutathione was impaired. This was also verified in the gene expression analysis, with a downregulation of genes involved in glucose metabolism. Additionally, both AKT1 KO and AKT2 KO had impaired fatty acid metabolism and folate biosynthesis, though possibly through different mechanisms.

However, genes were upregulated in the Wnt pathway and cell proliferation pathway which could oppose this effect. We therefore suggest that AKT inhibition should be combined with other drugs to attain the best effect.

Keyword [en]
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
URN: urn:nbn:se:uu:diva-222834OAI: oai:DiVA.org:uu-222834DiVA: diva2:712373
Swedish Cancer Society
Available from: 2014-04-14 Created: 2014-04-14 Last updated: 2014-06-30
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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 999
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
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)
Available from: 2014-05-15 Created: 2014-04-14 Last updated: 2014-08-15Bibliographically approved

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Häggblad Sahlberg, Sara
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