EGFR and Prion protein promote signaling via FOXO3a-KLF5 resulting in clinical resistance to platinum agents in colorectal cancer

Abstract

Epidermal growth factor receptor (EGFR) supports colorectal cancer progression via oncogenic signaling. Anti-EGFR therapy is being investigated as a clinical option for colorectal cancer, and an observed interaction between EGFR and Prion protein has been detected in neuronal cells. We hypothesized that PrP^C expression levels may regulate EGFR signaling and that detailed understanding of this signaling pathway may enable identification of resistance mechanisms and new actionable targets in colorectal cancer. We performed molecular pathway analysis following knockdown of PrP^C or inhibition of EGFR signaling via gefitinib to identify changes in expression of key signaling proteins that determine cellular sensitivity or resistance to cisplatin. Expression of these proteins was examined in matched primary and metastatic patient samples and was correlated for resistance to therapy and progression of disease. Utilizing three colorectal cancer cell lines, we observed a correlation between high expression of PrP^C and resistance to cisplatin. Investigation of molecular signaling in a resistant cell line revealed that PrP^C contributed to signaling via colocalization with EGFR, which could be overcome by targeting p38 mitogen-activated protein kinases (p38 MAPK). We revealed that the level of Krüppel-like factor 5 (KLF5), a target downstream of p38 MAPK, was predictive for cell line and patient response to platinum agents. Further, high KLF5 expression was observed in BRAF-mutant colorectal cancer. Our study indicates that the EGFR to KLF5 pathway is predictive of patient progression on platinum-based therapy.

Keywords: FOXO3a; Prion protein; cisplatin; colorectal cancer; signal transduction.

Figure 1

Prion protein associates with EGFR. (A) Western blot analysis of PrP^C expression in HT29, SW620, T84, and ‘normal‐like’ 293T cell lines. β‐Actin serves as a loading control. (B) Cisplatin dose–response curves of HT29, SW620, and T84 cell lines. Solid lines indicate normalized nonlinear fit and error bars ± SEM. (C) HT29, SW620, and T84 response to cisplatin treatment with or without PrP^C knockdown and p38 MAPK inhibitor. Cell viability was determined by MTS (% viability relative to DMSO control ± SEM and significance measured by two‐tailed Student's t‐test *P < 0.05). (D) Subcellular localization of PrP^C (green), EGFR (red), and nuclei‐stained DAPI (blue), scale bar 100 μm. (E) Fluorescence signal intensity of PrP^C and EGFR in HT29 cells, indicating colocalization. (F) Immunoprecipitation of PrP and EGFR by reciprocal antibodies. HSP70 probed for 1% of lysate input. (G) RNA‐seq analysis of 452 colon patient samples for correlation between PrP^C and EGFR expression levels and patient outcome (significance determined by logrank test). All data are the means of three independent biological replicates and error bars ± SEM. PRNPsi, PrP^C knockdown; p38i, p38 MAPK inhibitor.

Figure 2

Analysis of signaling downstream of PrP^C/EGFR by protein expression. (A) HT29, SW620, and T84 cell line viability in response to cisplatin treatment with or without gefitinib and p38 MAPK inhibitor. Cell viability determined by MTS (% viability relative to DMSO ± SEM and significance measured by two‐tailed Student's t‐test *P < 0.05) (B) Cell viability of HT29 cells in response to p38 MAPK inhibitor with or without PrP^C knockdown and gefitinib in the absence of cisplatin. Cell viability determined by MTS (% relative to DMSO ± SEM and significance measured by two‐tailed Student's t‐test *P < 0.05). (C) Protein expression analysis of the nuclear and cytosol fractions from HT29 cells from each treatment group. Histone H3 and α‐tubulin are used as loading controls for nuclear and cytosolic fractions, respectively. (D) Average densitometry of three independent isolates standardized to the controls and finally expressed as relative change to untreated cells. (E) Protein expression analysis of the nuclear and cytosol fractions from SW620 cells from each treatment group. Histone H3 and α‐tubulin are used as loading controls for nuclear and cytosolic fractions, respectively. (F) Average densitometry of two independent isolates standardized to the controls and finally expressed as relative change to untreated cells.

Figure 3

Knockdown of KLF5 sensitizes cells to cisplatin. (A) Relative mRNA expression levels of PRNP and KLF5 in response to indicated treatment HT29 cells and (B) SW620 cells. (Data normalized to GAPDH ± SEM and significance measured by two‐tailed Student's t‐test ***P < 0.001) (C) Targeted knockdown of KLF5 in HT29 and SW620 cells. (D) Targeted knockdown of PrP^C in HT29 and SW620 cells. (E) Cisplatin IC50 in HT29 cells and (F) SW620 cells following PrP^C, KLF5, or double knockdown determined by MTS. (Data compared to SCR control ± SEM and significance measured by two‐tailed Student's t‐test ***P < 0.001, *P < 0.05) SCR, nontargeting scrambled; PRNPsi, PrP^C knockdown; KLF5si, KLF5 knockdown; DKO, double (PrP^C and KLF5) knockdown. (G) A schematic showing the proposed EGFR signaling cascade in cisplatin‐sensitive and cisplatin‐resistant cells. The point of action of cisplatin, gefitinib, and p38 MAPK inhibitor is indicated.

Figure 4

The PrP^C/FOXO3a/KLF5 axis expression correlates with colorectal cancer patient progression and is a marker for outcome. (A) Representative images of negative and positive immunohistochemical staining of PrP^C, FOXO3a, and KLF5 from liver metastases, scale bar 100 μm. (B) Changes in positive staining percentages for PrP^C, FOXO3a, and KLF5 in 11 matched primary colorectal cancer and liver metastases. (C) Representative images of negative and positive KLF5 staining of BRAF‐mutant microsatellite stable (MSS) and BRAF‐mutant microsatellite unstable (MSI) cancers, scale bar 100 μm. (D) Comparison of PrP^C and KLF5 in BRAF‐wild‐type and BRAF‐mutant cancers, stratified by microsatellite instability (significance measured by two‐tailed Student's t‐test **P < 0.005). (E) Gene expression correlation with overall survival in 46 patients with aggressive Grade 3 colon cancer (significance determined by logrank test).