Simultaneous Inhibition of EGFR/VEGFR and Cyclooxygenase-2 Targets Stemness-Related Pathways in Colorectal Cancer Cells

<div><p>Despite the demonstrated benefits of anti-EGFR/VEGF targeted therapies in metastatic colorectal cancer (mCRC), many patients initially respond, but then show evidence of disease progression. New therapeutic strategies are needed to make the action of available drugs more efficient. Our study aimed to explore whether simultaneous targeting of EGFR/VEGF and cyclooxygenase-2 (COX-2) may aid the treatment and management of mCRC patients. The dual tyrosine kinase inhibitor AEE788 and celecoxib were used to inhibit EGFR/VEGFR and COX-2, respectively, in colorectal cancer cells. COX-2 inhibition with celecoxib augmented the antitumoral and antiangiogenic efficacy of AEE788, as indicated by the inhibition of cell proliferation, induction of apoptosis and G1 cell cycle arrest, down-regulation of VEGF production by cancer cells and reduction of cell migration. These effects were related with a blockade in the EGFR/VEGFR signaling axis. Notably, the combined AEE788/celecoxib treatment prevented β-catenin nuclear accumulation in tumor cells. This effect was associated with a significant downregulation of FOXM1 protein levels and an impairment in the interaction of this transcription factor with β-catenin, which is required for its nuclear localization. Furthermore, the combined treatment also reduced the expression of the stem cell markers Oct 3/4, Nanog, Sox-2 and Snail in cancer cells, and contributed to the diminution of the CSC subpopulation, as indicated by colonosphere formation assays. In conclusion, the combined treatment of AEE788 and celecoxib not only demonstrated enhanced anti-tumoral efficacy in colorectal cancer cells, but also reduced colon CSCs subpopulation by targeting stemness-related pathways. Therefore, the simultaneous targeting of EGFR/VEGF and COX-2 may aid in blocking mCRC progression and improve the efficacy of existing therapies in colorectal cancer.</p></div

Combined AEE788/celecoxib treatment downregulates stemness-related pathways in colorectal cancer cells.

<p>The expression of stem cell markers Oct 3/4, Nanog and Sox-2 was analyzed by western blot in total cell extracts of colon cancer cells after 6h of indicated treatments. The expression of -actin is included as loading control. The corresponding densitometric analysis is also shown. Data are means ± SEM of three independent experiments (*p <0.05, compared with the control; # p<0.05, compared with AEE788-treated cells).</p

AEE788 inhibits cell proliferation, induces apoptosis and alters cell cycle in colorectal cancer cells.

<p>A) Cell proliferation was evaluated after 72h of treatment with different doses of AEE788. B) Inhibition of cell proliferation by AEE788 was tested in cells growing in the presence of EGF (100 ng/ml). C) The fraction of apoptotic cells was estimated after 48 h of treatment with different doses of AEE788 of cells growing in the presence of EGF (100 ng/mL). D) Analysis of cell cycle was performed by flow cytometry after 48 h of treatment with different doses of AEE788 of cells growing in the presence of EGF (100 ng/mL). Data are means ± SEM of three independent experiments (*p <0.05, compared with the control).</p

Combined AEE788/Celecoxib impairs FOXM1- β-catenin interaction.

<p>To determine subcellular localization of β-catenin and FOXM1, both Caco-2 (A) and HCT-116 (B) cells were exposed to AEE788 (2.5 βM) and/or celecoxib (10 μM) for 6 h, stained for β-catenin (green) and FoxM (red) immunofluorescence, and counterstained with DAPI (blue). Merged images of β-catenin, FOXM1 and DAPI staining are also shown. Final magnification: X400. C) Pearson´s coefficient analysis was performed for the co-localization in cell nuclei of β-catenin and FOXM1. Data are means ± SEM of three independent experiments (*p <0.05, compared with the control). D) Cell extracts of Caco-2 cells after 6 h of the indicated treatments were subjected to IP using β-catenin antibody or control IgG, followed by IB with FOXM1 antibody.</p

AEE788 inhibits EGFR signaling in colorectal cancer cells.

<p>The phosphorylated and non-phosphorylated forms of EGFR, ERK 1/2 and Akt were detected by Western-blot using specific antibodies. Cells were grown in the absence or presence of EGF (100 ng/mL) and treated with AEE788 (2.5 µM) for 5, 10 or 15 min. The expression level of -actin was included as loading control.</p

Celecoxib intensifies the anti-angiogenic activity of AEE788.

<p>A) VEGF-A mRNA expression levels were assayed by real time RT-PCR in colorectal cancer cells growing in the presence of EGF (100 ng/ml) and exposed to the indicated treatments for 48 h. B) VEGFA165 levels were quantified by ELISA in the conditioned media collected from cells growing in the presence of EGF (100 ng/ml) and treated with AEE788 (2.5 µM) and/or celecoxib (10 μM) for 48h. Data are means ± SEM of three independent experiments (*p <0.05, compared with the control). C) The angiogenic activity of media conditioned by Caco-2 cells exposed for 24 h to the indicated treatments was evaluated using the endothelial tube assay as described under Material and Methods. Data are the total length of formed tubes in pixels (px), showing means ± SEM of three independent experiments (*p <0.05, compared with the control). D) Representative images of the formed interconnected networks after the treatment of endothelial cells with the indicated Caco-2 cells conditioned media. The extent of tube formation was quantified as indicated in the Material and Methods section. (Final magnification: X40, scale bar corresponds to 100 microns).</p

Combined AEE788/Celecoxib treatment downregulates FOXM1 protein levels in colorectal cancer cells.

<p>FOXM1 expression was analized by western-blot in cells were grown in the presence of EGF (100 ng/mL) and treated with AEE788 (2.5 µM) and/or celecoxib (10 μM) for 6h. The expression -actin is included as loading control. The corresponding densitometric analysis is also shown. Data are means ± SEM of three independent experiments (*p <0.05, compared with the control; # p<0.05, compared with AEE788-treated cells).</p

Combined AE788/Celecoxib treatment reduces the migratory capacity of colorectal cancer cells.

<p>A) Scratch wound healing assay was used to analyze the inhibition of cell migration in colorectal cancer cells treated for 24 h with AEE788 (2.5 µM) and/or celecoxib (10 µM). Data are means ± SEM of three independent experiments (*p <0.05, compared with the control; # p<0.05, compared with AEE788-treated cells). Final magnification: X100, scale bar corresponds to 100 microns. B) Representative images of scratched areas in confluent Caco-2 and HCT-116 cell layers. The yellow lines indicate the invasive front in the wound healing assay. Wound closure was photographed at 0h and 24 h after wounding. The scratched area at control (0 h) was arbitrarily assigned as 100%.</p

Combined AEE788/ Celecoxib treatment in colon cancer cells impairs colonosphere formation capability.

<p>Colon cancer cells were pre-treated with 2.5 µM AEE788 as a single agent or in combination with 10 µM celecoxib in the presence of 100 ng/mL EGF for 48h, and then cells were seeded at clonal density with serum free medium in low-adherence plates. After seven days, the number (A), size (B) and appearance (C) of formed colonospheres were evaluated by light microscopy. Spheres size was quantified in micrograph with the imaging software (Image J software). (Final magnification: X100, scale bar corresponds to 100 microns. Data are means ± SEM of three independent experiments (*p <0.05, compared with the control; # p<0.05, compared with AEE788-treated cells).</p

Inhibition of COX-2 enhances the antitumor efficacy of AEE788 in colorectal cancer cells.

<p>A) EGF-driven cell proliferation was assayed in cells growing in the presence of EGF (100 ng/ml) and in the presence or absence of AEE788 (2.5 μM), celecoxib (10 μM) and NS-398 (10 μM). B) Analysis of cell cycle was performed by flow cytometry after 48 h of treatment with AEE788 (2.5 μM) and/or celecoxib (10 μM) of cells growing in the presence of EGF (100 ng/mL). C) Expression levels of cyclooxygenase-2 (COX-2) were analyzed by western-blot in whole cell extracts form Caco-2 and HCT-116 cells. Expression of β-actin is included as loading control. D) The phosphorylated and non-phosphorylated forms of EGFR, ERK 1/2 and Akt were detected by Western-blot using specific antibodies. Cells were grown in the presence of EGF (100 ng/mL) and treated with AEE788 (2.5 µM) and/or celecoxib (10 μM) for 6h. The expression level of -actin was included as loading control. The corresponding densitometric analysis is also shown. Data are means ± SEM of three independent experiments (*p <0.05, compared with the control; # p<0.05, compared with AEE788-treated cells).</p