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. 2017 Jan 1;23(1):159-170.
doi: 10.1158/1078-0432.CCR-16-0709. Epub 2016 Jul 11.

Dysregulation of EGFR Pathway in EphA2 Cell Subpopulation Significantly Associates with Poor Prognosis in Colorectal Cancer

Mariangela De Robertis  1 , Luisa Loiacono  2   3 , Caterina Fusilli  4 , Maria Luana Poeta  5 , Tommaso Mazza  4 , Massimo Sanchez  6 , Luigi Marchionni  7 , Emanuela Signori  8 , Giuseppe Lamorte  9 , Angelo Luigi Vescovi  9 , Jesus Garcia-Foncillas  10 , Vito Michele Fazio  1   3
Affiliations

Dysregulation of EGFR Pathway in EphA2 Cell Subpopulation Significantly Associates with Poor Prognosis in Colorectal Cancer

Mariangela De Robertis et al. Clin Cancer Res. .

Abstract

Purpose: EphA2 receptor is involved in multiple cross-talks with other cellular networks, including EGFR, FAK, and VEGF pathways, with which it collaborates to stimulate cell migration, invasion, and metastasis. Colorectal cancer (CRC) EphA2 overexpression has also been correlated to stem-like properties of cells and tumor malignancy. We investigated the molecular cross-talk and miRNAs modulation of the EphA2 and EGFR pathways. We also explored the role of EphA2/EGFR pathway mediators as prognostic factors or predictors of cetuximab benefit in patients with CRC.

Experimental design: Gene expression analysis was performed in EphA2high cells isolated from CRC of the AOM/DSS murine model by FACS-assisted procedures. Six independent cohorts of patients were stratified by EphA2 expression to determine the potential prognostic role of a EphA2/EGFR signature and its effect on cetuximab treatment response.

Results: We identified a gene expression pattern (EphA2, Efna1, Egfr, Ptpn12, and Atf2) reflecting the activation of EphA2 and EGFR pathways and a coherent dysregulation of mir-26b and mir-200a. Such a pattern showed prognostic significance in patients with stage I-III CRC, in both univariate and multivariate analysis. In patients with stage IV and WT KRAS, EphA2/Efna1/Egfr gene expression status was significantly associated with poor response to cetuximab treatment. Furthermore, EphA2 and EGFR overexpression showed a combined effect relative to cetuximab resistance, independently from KRAS mutation status.

Conclusions: These results suggest that EphA2/Efna1/Egfr genes, linked to a possible control by miR-200a and miR-26b, could be proposed as novel CRC prognostic biomarkers. Moreover, EphA2 could be linked to a mechanism of resistance to cetuximab alternative to KRAS mutations. Clin Cancer Res; 23(1); 159-70. ©2016 AACR.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1
Isolation of mouse colorectal cell populations based on EphA2 and EphB2 expression. (A) IHC analysis on normal colorectal tissue of control untreated mice demonstrated maximum EphA2 and EphB2 expression in crypt apical columnar cells (white arrowhead) and basal crypt compartment (black arrowhead), respectively; adenocarcinoma shows a diffuse staining for both EphA2 and EphB2 (20× and 40× magnification). (B) Flow cytometry of crypt cells stained for EphA2 revealed an increase of EphA2high cell subpopulation in adenocarcinoma with respect to normal mucosa. EphB2high cells were poorly represented in normal mucosa and colon adenocarcinoma. (C) Representative cell sorting strategy. EphA2high and EphA2low cells as well as EphB2high and EphB2low subpopulations were sorted after gating for CD45- and EpCAM+ staining to ensure epithelial identity. Fluorescence Minus One (FMO) control stain strategy was used to accurately identify EphA2 and EphB2 expressing cells in the fully stained sample.
Figure 2
Figure 2
(A) Q-PCR analysis of differentiation (Krt20) and stem cell markers (Lgr5, Ascl2) in EphA2high/low and EphB2high/low cell subpopulations purified from murine normal colon and colorectal adenocarcinoma. Data are represented as mean +/− SD. Statistically significant differences were calculated using Student’s T-test: *** p<0.0001; ** p<0.001; * p<0.01. (B) IHC analysis of Krt20 and Lgr5 protein in normal murine colon. Left panels: cells on the top of the crypt were strongly stained for Krt20. Right panels: cells at the crypt bottom were strongly stained for Lgr5 (20× and 40× magnification).
Figure 3
Figure 3
Q-PCR analysis of EGFR signaling effectors in EphA2 cell subpopulations of murine CRC. Data are represented as mean +/− SD. Statistically significant differences were calculated using Student’s t-test: *** p<0.0001; ** p<0.001; * p<0.01. Gene expression levels in EphA2high and EphA2low cell subpopulations of (A) normal mucosa and (B) adenocarcinoma. (C) Gene expression levels in EphA2high subpopulation of adenocarcinoma and EphA2high subpopulation of normal colic mucosa. (D) Schematic representation of the dysregulation of EphA2/EGFR pathways crosstalk in adenocarcinoma EphA2high cell.
Figure 4
Figure 4
Kaplan-Meier survival curves of (A) EphA2high (dashed line) versus EphA2low (solid line) for cohort 1, 3, 4, 5 and 6 (B) Efna1high (dashed line) versus Efna1low (solid line) for cohort 2, 4 and 5. (C) Analysis of Efna1 conducted only for patients belonging to EphA2high group for the same cohorts of B. (D) Kaplan-Meier survival curves on TCGA dataset of mir-200ahigh (dashed line) versus mir-200alow (solid line) and mir-26bhigh (dashed line) versus mir-26blow (solid line). Expression value thresholds were determined through maxstat R package. P-values were calculated using log-rank tests. Tick marks represent censored data.
Figure 5
Figure 5
(A) Kaplan-Meier survival curves of EphA2, Efna1 and EGFR for cohort 5. Survival curves of EphA2high (dashed line) versus EphA2low (solid line), Efna1high (dashed line) versus Efna1low (solid line) and EGFRhigh (dashed line) versus EGFRlow (solid line) for all patients of the cohort. P-values were calculated using log-rank tests. Expression value thresholds for determining high and low groups were determined through maxstat R package. (B) Analysis of Efna1 and EGFR conducted only for patients belonging to EphA2high group. EphA2high group was determined with EphA2 median expression threshold. (C) Survival curves of EphA2 and Efna1 for patients with WT KRAS. (D) Survival curves of EphA2 and Efna1 for patients with mutant KRAS. P-values were calculated using log-rank tests.
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