Sonic hedgehog pathway activation is associated with cetuximab resistance and EPHB3 receptor induction in colorectal cancer

Abstract

A major problem of colorectal cancer (CRC) targeted therapies is relapse caused by drug resistance. In most cases of CRC, patients develop resistance to anticancer drugs. Cetuximab does not show many of the side effects of other anticancer drugs and improves the survival of patients with metastatic CRC. However, the molecular mechanism of cetuximab resistance is not fully understood. Methods: EPHB3-mediated cetuximab resistance was confirmed by in vitro western blotting, colony-forming assays, WST-1 colorimetric assay, and in vivo xenograft models (n = 7 per group). RNA-seq analysis and receptor tyrosine kinase assays were performed to identify the cetuximab resistance mechanism of EPHB3. All statistical tests were two-sided. Results: The expression of EFNB3, which upregulates the EPHB3 receptor, was shown to be increased via microarray analysis. When resistance to cetuximab was acquired, EPHB3 protein levels increased. Hedgehog signaling, cancer stemness, and epithelial-mesenchymal transition signaling proteins were also increased in the cetuximab-resistant human colon cancer cell line SW48R. Despite cells acquiring resistance to cetuximab, STAT3 was still responsive to EGF and cetuximab treatment. Moreover, inhibition of EPHB3 was associated with decreased STAT3 activity. Co-immunoprecipitation confirmed that EGFR and EPHB3 bind to each other and this binding increases upon resistance acquisition, suggesting that STAT3 is activated by the binding between EGFR and EPHB3. Protein levels of GLI-1, SOX2, and Vimentin, which are affected by STAT3, also increased. Similar results were obtained in samples from patients with CRC. Conclusion: EPHB3 expression is associated with anticancer drug resistance.

Keywords: Cetuximab Resistance; EPHB3; GLI-1; colorectal cancer.

Figure 1

Effects of cetuximab on human CRC cell lines with acquired resistance to cetuximab, including ephrin-EPHB3 signaling and mTOR activation. (A) SW48 and SW48R cells were treated with increasing concentrations of cetuximab (5, 10, 15, 20 μg/mL) for 48 h and 72 h after overnight 2% FBS starvation. Cell viability was determined using a WST-1 assay. (B) SW48R cells showed improved cell proliferation for 5 days compared with SW48 cells. (C) SW48 and SW48R cells were treated with cetuximab (5, 10, 20 μg/mL). After two weeks, cells were stained with crystal violet and photographed. (D) Heat map showing the results of gene set enrichment analysis of genes significantly modified by cetuximab (10 μg/mL) in SW48 cells for 3 months. Plots of the mean gene expression values of leading-edge genes for each gene set. Lower levels of expression are displayed in green and higher levels in red. Gene sets representing differentially enriched pathways are grouped. (E and F) Enrichment plots of representative EPHB3 and mTOR gene sets. The relative gene positions of the gene sets are indicated by vertical lines below the graphs, which present the enrichment scores of individual genes. Lines clustered to the left represent higher-ranked genes in the list. Bottom plot shows the rank matrix of these genes. The position of leading-edge genes suggests a positive correlation between cetuximab treatment and the EGFR pathway. (G) Protein was collected and fractionated by SDS-PAGE, followed by immunoblotting for the indicated proteins. β-actin was used as a loading control. Data are expressed as the means of three independent experiments. **P < 0.01, *P < 0.05.

Figure 2

Cetuximab induced stemness, hedgehog signaling, and EMT in CRC cells. (A) Western blotting was performed to detect levels of stemness markers p-STAT3, Nanog, OCT4, SOX2, and EpCAM in SW48 and SW48R cells. β-actin was used as a loading control. (B) Western blotting was performed to detect levels of hedgehog markers GLI-1, GLI-2, GLI-3, SHH, SMO, and PTCH in SW48 and SW48R cells. (C) Western blotting was performed to detect levels of EMT markers E-Cadherin, N-Cadherin, Vimentin, and Snail in SW48 and SW48R cells. (D) SW48 and SW48R cells were treated with increasing concentrations of cetuximab (5, 10, and 20 μg/mL) for 24h, with EGF, after overnight 2% FBS starvation. Protein was collected and fractionated by SDS-PAGE, followed by immunoblotting for the indicated proteins. β-actin was used as a loading control. Data are expressed as the means of three independent experiments. (E) STAT3 was silenced in SW48R cells with STAT3 siRNA. The levels of GLI-1, p- STAT3, STAT3, SOX2, and Vimentin were detected by western blotting. (F) GLI-1 was silenced in SW48R cells with GLI-1 siRNA. The levels of GLI-1, p- STAT3, STAT3, SOX2, and Vimentin were detected by western blotting. β-actin was used as a loading control. Data are expressed as the means of three independent experiments. FBS: fetal bovine serum; EMT: epithelial-mesenchymal transition; siRNA: short interfering RNA.

Figure 3

Ephrin-EPHB3 receptor signaling is upregulated in cetuximab-resistant cells. (A) Schematic representation of the proposed model related to cetuximab-induced EPHB3 and hedgehog activation. (B) Characterization of the expression of EPHB3 family members in EFNB3-treated SW48 parent cells. Proteins were collected and fractionated by SDS-PAGE followed by immunoblotting for the indicated proteins. β-actin was used as a loading control. (C) SW48 cells were treated with increasing concentrations of cetuximab (5, 10, 15, and 20 μg/mL) for 24 h and 48 h, with EFNB3, after overnight 2% FBS starvation. Cell viability was determined by the WST-1 assay. (D) SW48 and SW48R cells were treated with increasing concentrations of cetuximab (5, 10, and 20 μg/mL) for 24 h with EGF after overnight 2% FBS starvation. Stimulation was with EGF (10 ng/mL) for 30 min. Proteins were collected and fractionated by SDS-PAGE followed by immunoblotting for the indicated proteins. β-actin was used as a loading control. Data are expressed as the means of three independent experiments. (E) The immunofluorescence of EPHB3 was detected by confocal laser-scanning microscopy (original magnification, 40×). Scale bar: 10 µm. (F) Cetuximab treatment in cetuximab-sensitive cell line (KRAS wild-type (WT)) and cetuximab-resistant cell line (KRAS mutant-type (MT)) for 5 months. Proteins were collected and fractionated by SDS-PAGE, followed by immunoblotting for the indicated proteins. β-actin was used as a loading control. Data are expressed as the means of three independent experiments. (G) SW48 cells were treated with various anti-cancer drugs (cetuximab (EGFR), AZD4547 (FGFR), imatinib (PDGFR), and bevacizumab (VEGFR)) for 3 months. Proteins were collected and fractionated by SDS‑PAGE followed by immunoblotting for the indicated proteins. β-actin was used as a loading control. Data are expressed as the means of three independent experiments. FBS: fetal bovine serum. *P < 0.05.

Figure 4

Blockade of EPHB3 could effectively inhibit the proliferation and induction of apoptosis of SW48R cells. (A) The levels of EPHB3 signaling, hedgehog signaling, and cell growth signaling were examined using western blotting after treatment with an EPHB3 inhibitor (20 μM) for the indicated times. Total cell protein extracts were subjected to immunoblotting with the indicated antibodies, as described in the Materials and Methods. β-actin was used as a loading control. (B) Combinatorial treatment with cetuximab and the EPHB3 inhibitor led to loss of EPHB3 expression in SW48 and SW48R cells. The levels of c-PARP, STAT3, p- STAT3 and GLI-1 were detected by western blotting. (C) Knockdown of STAT3 cells treated with cetuximab led to loss of STAT3 expression in SW48 and SW48R cells. The levels of c-PARP, STAT3, p- STAT3 and GLI-1 were detected by western blotting. (D) SW48R cells treated with the EPHB3 inhibitor were stained with annexin V and propidium iodide (PI), and examined by FACS analysis. (E) SW48R cells transfected with control siRNA or STAT3 siRNA were stained with annexin V and PI, and examined by FACS analysis. (F) SW48 and SW48R cells were treated with cetuximab or the EPHB3 inhibitor for 24 h. The WST-1 assay was used to evaluate the effects of the EPHB3 inhibitor on proliferation. (G) SW48 and SW48R cells transfected with control or STAT3 siRNA were treated with cetuximab for 24 h. The WST-1 assay was used to evaluate the effects of STAT3 expression on proliferation. *P < 0.05. siRNA: short interfering RNA; PI: propidium iodide; FACS: fluorescence activated cell sorting.

Figure 5

EGF treatment of SW48 cells renders these cells resistant to cetuximab, and EGF induces the EGFR-EPHB3 interaction. (A) EGFR domain (Red) and EPHB3 domain (green). (B) EGFR displayed increased association with EPHB3 in SW48R cells compared with SW48 cells. Cells were harvested and EGFR or EPHB3 were immunoprecipitated with anti-rabbit EGFR antibodies or anti-rabbit EPHB3 antibodies. The immunoprecipitated complexes were fractionated by SDS-PAGE and immunoblotting was performed on the indicated proteins. (C) SW48 and SW48R cells were immunostained for EGFR (green) or EPHB3 (red) and examined using confocal microscopy. Scale bar, 10 μm. (D) Quantification of the co-localization in fig C. Error bars represent the mean ± SEM from each cell (*P < 0.05). (E) Level of extracellular EGF protein expression by EPHB3 in SW48R cells compared with that in SW48 cells was measured using ELISA. Results are represented by the mean values of EGF concentrations, and error bars represent the SEM from three separate experiments. (F) Western blotting analysis of protein expression in SW48 and SW48R cells treated with EGF (10 ng/mL) for 30 min. The proteins were collected and fractionated by SDS-PAGE and immunoblotting was performed for the indicated proteins. (G) The protein levels of p-EGFR family members were evaluated using western blotting after treatment with the EPHB3 inhibitor (20 μM) at the indicated times. β-actin was used as a loading control. Data are expressed as the means of three independent experiments. ELISA: enzyme-linked immunosorbent assay.

Figure 6

Combination of cetuximab and EPHB3 inhibitor treatment of SW48R tumor cells leads to growth delay in vivo. (A and B) SW48R cells were implanted subcutaneously into nude mice, and then tumor growth was examined by measuring the tumor volume after 3 weeks of treatment with cetuximab (10 mg/kg), the EPHB3 inhibitor (0.1 mg/kg), or the combination of cetuximab and the EPHB3 inhibitor (every 2 days; n = 7). (C) Line graph showing the tumor volume (mm³) in SW48R cell tumor-bearing mice treated with PBS alone, cetuximab alone, the EPHB3 inhibitor alone, or a combination of cetuximab and the EPHB3 inhibitor, from day 0 to day 34. Error bars represent the mean ± SD from 5 mice. For statistical analysis, Student's t‐test (two‐sided, paired) was used. **P < 0.01. (D) Tumors were examined using the TUNEL assay, and DAPI was used to visualize the nuclei. (E) The percentage of TUNEL-positive cells was determined and plotted as a histogram. **P < 0.01. (F) The inhibition of EGFR and EPHB3 expression in SW48R tumors after combinatorial treatment is consistent with reduced proliferation and increased apoptosis. SW48R tumor samples after treatment with cetuximab, the EPHB3 inhibitor, or the combination of cetuximab and EPHB3 inhibitor in vivo were prepared and analyzed for EGFR and EPHB3 by Immunofluorescence staining. Images were measured by taking the average staining intensity quantified from three tumors per treatment group (three images/tumor, n = 7). Magnification 100×. PBS: phosphate buffered saline; TUNEL: terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling; DAPI: 4, 6-diamidino-2-phenylindole.