Characteristics of both Cohort 1 and Cohort 2 groups of PDAC patients.

<p>Characteristics of both Cohort 1 and Cohort 2 groups of PDAC patients.</p

ROC Curves for the 5 genes commonly expressed: <i>FAMI3</i>, <i>IRAK3</i>, <i>DENND2D</i>, <i>PLBD1</i> and <i>AGPAT9</i>.

<p>Curves are provided for both Illumina and Affymetrix individual analyses as well as our integrative meta-analysis. The Area Under the Curve (AUC) metrics are also provided for each curve.</p

Coincident genes in the three analyzes: Affymetrix, Illumina and integrated meta-analysis.

<p>Coincident genes in the three analyzes: Affymetrix, Illumina and integrated meta-analysis.</p

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

Comparison of individual analysis by technology with integrated analysis.

<p><b>a</b> Coincident genes in the three analyzes: Affymetrix, Illumina and integrated meta-analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194844#pone.0194844.t002" target="_blank">Table 2</a>). <b>b</b> Remaining differentially expressed genes in individual Illumina and the integrative meta-analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194844#pone.0194844.s006" target="_blank">S1 Table</a>). <b>c</b> Remaining differentially expressed genes in individual Affymetrix and the integrative meta-analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194844#pone.0194844.s007" target="_blank">S2 Table</a>). <b>d</b> Differentially expressed genes in the integrative meta-analysis but not in individual analysis (gained genes) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194844#pone.0194844.s008" target="_blank">S3 Table</a>).</p

Shared Gene Ontology (GO) terms after the gene enrichment analysis applied over the 28 gained genes.

<p>The Kolmogorov-Smirnov statistical test was performed to determine their significance (<i>p</i>-value < 0.05).</p

CoCl₂, a Mimic of Hypoxia, Induces Formation of Polyploid Giant Cells with Stem Characteristics in Colon Cancer

<div><p>The induction of polyploidy is considered the reproductive end of cells, but there is evidence that polyploid giant cancer cells (PGCCs) contribute to cell repopulation during tumor relapse. However, the role of these cells in the development, progression and response to therapy in colon cancer remains undefined. Therefore, the main objective of this study was to investigate the generation of PGCCs in colon cancer cells and identify mechanisms of formation. Treatment of HCT-116 and Caco-2 colon cancer cells with the hypoxia mimic CoCl₂ induced the formation of cells with larger cell and nuclear size (PGCCs), while the cells with normal morphology were selectively eliminated. Cytometric analysis showed that CoCl₂ treatment induced G2 cell cycle arrest and the generation of a polyploid cell subpopulation with increased cellular DNA content. Polyploidy of hypoxia-induced PGCCs was confirmed by FISH analysis. Furthermore, CoCl₂ treatment effectively induced the stabilization of HIF-1α, the differential expression of a truncated form of p53 (p47) and decreased levels of cyclin D1, indicating molecular mechanisms associated with cell cycle arrest at G2. Generation of PGCCs also contributed to expansion of a cell subpopulation with cancer stem cells (CSCs) characteristics, as indicated by colonosphere formation assays, and enhanced chemoresistance to 5-fluorouracil and oxaliplatin. In conclusion, the pharmacological induction of hypoxia in colon cancer cells causes the formation of PGCCs, the expansion of a cell subpopulation with CSC characteristics and chemoresistance. The molecular mechanisms involved, including the stabilization of HIF-1 α, the involvement of p53/p47 isoform and cell cycle arrest at G2, suggest novel targets to prevent tumor relapse and treatment failure in colon cancer.</p></div

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

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

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