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Research Article
Open Access

ILK Expression in Colorectal Cancer Is Associated with EMT, Cancer Stem Cell Markers and Chemoresistance

DIMITRIOS TSOUMAS, SOFIA NIKOU, EFSTATHIA GIANNOPOULOU, SPYRIDON CHAMPERIS TSANIRAS, CHAIDO SIRINIAN, IOANNIS MAROULIS, STAVROS TARAVIRAS, VASSILIKI ZOLOTA, HARALABOS P. KALOFONOS and VASILIKI BRAVOU
Cancer Genomics & Proteomics March 2018, 15 (2) 127-141;
DIMITRIOS TSOUMAS
1Department of Anatomy-Histology-Embryology, Medical School, University of Patras, Patras, Greece
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SOFIA NIKOU
1Department of Anatomy-Histology-Embryology, Medical School, University of Patras, Patras, Greece
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EFSTATHIA GIANNOPOULOU
2Clinical Oncology Laboratory, University of Patras Medical School, Patras, Greece
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SPYRIDON CHAMPERIS TSANIRAS
3Department of Physiology, Medical School, University of Patras, Patras, Greece
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CHAIDO SIRINIAN
2Clinical Oncology Laboratory, University of Patras Medical School, Patras, Greece
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IOANNIS MAROULIS
4Department of Surgery, University of Patras Medical School, Patras, Greece
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STAVROS TARAVIRAS
3Department of Physiology, Medical School, University of Patras, Patras, Greece
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VASSILIKI ZOLOTA
5Department of Pathology, University of Patras Medical School, Patras, Greece
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HARALABOS P. KALOFONOS
2Clinical Oncology Laboratory, University of Patras Medical School, Patras, Greece
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VASILIKI BRAVOU
1Department of Anatomy-Histology-Embryology, Medical School, University of Patras, Patras, Greece
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  • For correspondence: vibra@upatras.gr
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    Figure 1.

    Immunohistochemical expression of ILK, EMT and CSC markers in human CRC. Representative cases of human CRC with strong expression of ILK (A), nuclear and cytoplasmic expression of β-catenin (B), loss of membranous expression of E-cadherin (C), strong nuclear expression of ZEB (D), strong expression (mainly cytoplasmic) of Snail (E), membranous expression of CD44 (F), strong expression (mainly cytoplasmic) of Lgr5 (G) and strong expression (mainly cytoplasmic) of CD133 (H). Bars correspond to 50 μm.

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    Figure 2.

    ILK co-localizes with ZEB and Lgr5 in human CRC by double immunofluorescence. ILK expression is shown in red, ZEB and Lgr5 expression is shown in green. Nuclei are stained with Hoechst (blue). Merged photos of red and green (ILK-ZEB and ILK-Lgr5) show co-localization in tumor cells.

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    Figure 3.

    Expression of ILK, E-cadherin, ZEB and CD44 in metastatic CRC is associated with chemotherapy response. Immunohistochemical expression of ILK (A, B), E-cadherin (C, D), ZEB (E, F) and CD44 (G, H) is shown in representative cases of responders (left column) and non-responders (right column). Non-responders to chemotherapy show higher expression of ILK (B), ZEB (F) and CD44 (H) and decreased expression of E-cadherin (D) compared to responders. Bars correspond to 50 μm.

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    Figure 4.

    Pharmacological inhibition of ILK in HT29 cells reduces acquired resistance to 5-FU and oxaliplatin and lowers levels of p-Akt. (A) Results from MTT assays. Logarithmic dose-response (growth) curves, with IC50 values indicated, showing the effect of QLT0267 on parental HT29 and resistant 5-FUR (i) or OxalR cells (ii), as well as the effect of 5-FU (iii) or oxaliplatin (iv) on resistant cells (5FUR and OxalR respectively) pretreated with QLT (5FURQLT and OxalRQLT respectively). QLT0267 results in 3- to 4-fold greater inhibition of cell proliferation of 5FUR (IC50=26 μM) (i) and OxalR cells (IC50=34 μM) (ii) compared to parental HT29 cells (IC50>100μM. Pre-treatment with QLT0267 increases sensitivity to growth inhibition by 5-FU and oxaliplatin. (B) Protein expression by immunoblotting of ILK and p-Akt in HT29 colon cancer cells resistant to 5-FU (5-FUR) and oxaliplatin (OxalR), as well as, in resistant cells treated with ILK inhibitor QLT0267 (5-FUR+QLT and OxalR+QLT). Representative results of three independent experiments are shown.

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    Figure 5.

    Pharmacological inhibition of ILK in HT29 cells resistant to 5-FU inhibits expression of EMT and CSC markers. (A) EMT-consistent phenotypic changes upon 5-FU resistance. Spindle-shape morphology and pseudopodia formation are noted in resistant to 5-FU (5-FUR), but not in parental HT29 cells. (B) Protein expression by immunoblotting of EMT markers (β-catenin, E-cadherin, Vimentin and Snail) and CSC markers (Lgr5) in HT29, 5-FUR cells and 5-FUR cells treated with QLT0267 (5-FUR+QLT). 5-FUR cells express higher protein levels of active β-catenin, lower levels of E-cadherin and higher levels of Vimentin, Snail and Lgr5 compared to parental HT29 cells, while QLT0267 treatment (5-FUR+QLT) reverses these effects. Representative results of three independent experiments are shown.

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    Figure 6.
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    Figure 6.

    Immunofluorescence analysis of EMT markers in HT29, 5-FUR cells and 5-FUR cells pre-treated with QLT0267 (5-FUR+QLT). Decreased expression of E-cadherin and increased expression of vimentin, Snail and ZEB can be seen in 5-FU resistant cells (5-FUR) compared to HT29 cells while treatment of resistant cells with QLT0267 (5-FUR+QLT) results in increased expression of E-cadherin and decreased expression of Vimentin, Snail and ZEB. Nuclei are stained with Hoechstain. Representative results of three independent experiments are shown.

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Cancer Genomics & Proteomics
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March-April 2018
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ILK Expression in Colorectal Cancer Is Associated with EMT, Cancer Stem Cell Markers and Chemoresistance
DIMITRIOS TSOUMAS, SOFIA NIKOU, EFSTATHIA GIANNOPOULOU, SPYRIDON CHAMPERIS TSANIRAS, CHAIDO SIRINIAN, IOANNIS MAROULIS, STAVROS TARAVIRAS, VASSILIKI ZOLOTA, HARALABOS P. KALOFONOS, VASILIKI BRAVOU
Cancer Genomics & Proteomics Mar 2018, 15 (2) 127-141;

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ILK Expression in Colorectal Cancer Is Associated with EMT, Cancer Stem Cell Markers and Chemoresistance
DIMITRIOS TSOUMAS, SOFIA NIKOU, EFSTATHIA GIANNOPOULOU, SPYRIDON CHAMPERIS TSANIRAS, CHAIDO SIRINIAN, IOANNIS MAROULIS, STAVROS TARAVIRAS, VASSILIKI ZOLOTA, HARALABOS P. KALOFONOS, VASILIKI BRAVOU
Cancer Genomics & Proteomics Mar 2018, 15 (2) 127-141;
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Keywords

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  • cancer stem cells
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