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. 2016 May 24;7(21):31350-60.
doi: 10.18632/oncotarget.8912.

CYB5R1 Links Epithelial-Mesenchymal Transition and Poor Prognosis in Colorectal Cancer

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Free PMC article

CYB5R1 Links Epithelial-Mesenchymal Transition and Poor Prognosis in Colorectal Cancer

Christine Woischke et al. Oncotarget. .
Free PMC article

Abstract

Colorectal cancers show significant tumor cell heterogeneity within the same core genetic background. Epithelial-mesenchymal transition (EMT) is an important functional aspect of this heterogeneity and hallmark of colorectal cancer progression. Here, we identify CYB5R1, an enzyme involved in oxidative stress protection and drug metabolism, as an indicator of EMT in colon cancer. We demonstrate high CYB5R1 expression in colorectal cancer cells undergoing EMT at the infiltrative tumor edge and reveal an extraordinarily strong association of CYB5R1 expression with two core EMT gene expression signatures in a large independent colon cancer data set from The Cancer Genome Atlas (TCGA). Furthermore, we demonstrate that CYB5R1 is required for an infiltrative tumor cell phenotype, and robustly linked with poor prognosis in colorectal cancer. Our findings have important implications for colon cancer cells undergoing EMT and may be exploited for diagnostic and therapeutic purposes.

Keywords: CYB5R1; EMT; colorectal cancer; drug metabolism; survival.

Conflict of interest statement

No conflicts of interest.

Figures

Figure 1
Figure 1. Immunostaining of CYB5R1 in colorectal cancer
Assessment of CYB5R1 staining in a collection of 221 primary colorectal cancers. Tumors were assigned scores from 0 (no CYB5R1 staining) to 3 (strong CYB5R1 staining in most tumor cells). Arrows indicate CYB5R1 staining in tumor cells at the infiltrative edge. Images in lower panels show higher magnifications of the areas boxed in upper panels.
Figure 2
Figure 2. Confocal immune fluorescence for CYB5R1
Staining of individual colorectal cancer cases indicates loss of E-Cadherin (CDH1) expression in tumor cells with strong CYB5R1 expression (upper panels), and co-localization of CYB5R1 with Laminin gamma 2 (LAMC2, lower panels). Arrowheads indicate infiltrative colon cancer cells at the leading tumor edge. Asterisks indicate tumor cells with glandular differentiation. Right panels show composites of left and mid panels.
Figure 3
Figure 3. CYB5R1 is linked to EMT in gene expression data of colon cancers from the TCGA
A. Gene Set Enrichment Analyses for genes ranked by Pearson correlation (Pearson r) of expression to CYB5R1 indicates enrichment for two core EMT gene signatures [17, 18]. ES=enrichment score. p < 0.001. B. Heat map indicates clustering and positive correlation of CYB5R1 expression with colon cancer relevant EMT markers and negative correlation with CDH1. Colors indicate Pearson r from −1 (blue) to 1 (red).
Figure 4
Figure 4. CYB5R1 depletion decreases migration and invasion of colon cancer cells
A. Immunoblotting for indicated proteins on whole cell lysates of DLD-1 and HCT116 colon cancer cells harvested 48 h after transfection with CYB5R1 or control (Ctrl) siRNA. B–C. Representative micrographs (left panels) and quantification (right panels) of migrated or invaded DLD-1 (B) and HCT116 (C) colon cancer cells in transwell assays. Data are mean ± SD, n ≥ 3, p-values are t test results.
Figure 5
Figure 5. CYB5R1 indicates poor survival in colorectal cancer
Kaplan-Meier plots for different CYB5R1 expression scores for tumor specific survival A. and disease free survival B. indicate significant poorer outcome with increasing CYB5R1 expression. Results for dichotomal CYB5R1 expression (negative and positive) are shown in C. and D. Significance p-values indicate log-rank test results. Ratios on curves indicate the number of events over the number of patients per group.
Figure 6
Figure 6. CYB5R1 mRNA expression and survival in colon cancer data from the TCGA
A. ROC curve for determining best discrimination thresholds of CYB5R1 mRNA reads for survival prediction. Arrow indicates chosen value for binary classification. AUC=Area under curve. B. Kaplan-Meier statistics for binary (low and high) classified CYB5R1 expression indicate shorter tumor specific survival for CYB5R1 high. Significance p-values indicate log-rank test results. Ratios on curves indicate the number of events over the number of patients per group. C, D. Cases with high CYB5R1 expression are significantly (t-test) more frequent among cases with lymph node metastasis (N+, C) and distant metastasis (M1, D), when compared to cases with low CYB5R1 expression.

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