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. 2019 Jun 13;11(6):821.
doi: 10.3390/cancers11060821.

Hedgehog Pathway as a Potential Intervention Target in Esophageal Cancer

Da Wang  1   2   3 , Peter W Nagle  4   5   6 , Helena H Wang  7   8   9 , Justin K Smit  10 , Hette Faber  11   12 , Mirjam Baanstra  13   14 , Arend Karrenbeld  15 , Roland K Chiu  16   17 , John Th M Plukker  18 , Robert P Coppes  19   20
Affiliations

Hedgehog Pathway as a Potential Intervention Target in Esophageal Cancer

Da Wang et al. Cancers (Basel). .

Abstract

Esophageal cancer (EC) is an aggressive disease with a poor prognosis. Treatment resistance is a major challenge in successful anti-cancer therapy. Pathological complete response after neoadjuvant chemoradiation (nCRT) is low, thus requiring therapy optimization. The Hedgehog (HH) pathway has been implicated in therapy resistance, as well as in cancer stemness. This article focusses on the HH pathway as a putative target in the treatment of EC. Immunohistochemistry on HH members was applied to EC patient material followed by modulation of 3D-EC cell cultures, fluorescence-activated cell sorting (FACS), and gene expression analysis after HH pathway modulation. Sonic Hedgehog (SHH) and its receptor Patched1 (PTCH1) were significantly enriched in EC resection material of patients with microresidual disease (mRD) after receiving nCRT, compared to the control group. Stimulation with SHH resulted in an up-regulation of cancer stemness in EC sphere cultures, as indicated by increased sphere formation after sorting for CD44+/CD24- EC cancer stem-like cell (CSC) population. On the contrary, inhibiting this pathway with vismodegib led to a decrease in cancer stemness and both radiation and carboplatin resistance. Our results strengthen the role of the HH pathway in chemoradiotherapy resistance. These findings suggest that targeting the HH pathway could be an attractive approach to control CSCs.

Keywords: Esophageal cancer; Hedgehog pathway; cancer stem cells; treatment resistance.

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Conflict of interest statement

The authors declare that there is no conflict of interest.

Figures

Figure 1
Figure 1
PTCH1 and SHH are up-regulated in mRD patients compared to surgery alone patients (S). (A) Representative samples of low intensity PTCH1 and weakly positive SHH expression (respectively upper left and lower left), and high intensity PTCH1 and strong positive SHH expression (respectively upper right and lower right). (B) Comparison of PTCH1 (p = 0.04) and SHH (p = 0.04) IHC expression between mRD after neoadjuvant CRT resection specimens (N = 16) and S specimens (N = 32). Error bars represent standard error of the mean (SEM), *p < 0.05.
Figure 2
Figure 2
Enhancement of sphere formation capacity by sorting for CD44+/CD24−. (A) Representative FACS plots of OE21 and OE33 stained with CD24 FITC and CD44 PE. (B) Representative images of spheres. Bar indicates 100 µm. (C) Quantification of spheres shown in (B). OE21 CD44+/CD24− vs. CD44+/CD24+ (p = 0.01) and OE33 CD44+/CD24− vs. CD44+/CD24+ (p = 0.02, N = 3) spheres after five days of culture. Error bars represent standard deviation, *p < 0.05.
Figure 3
Figure 3
PTCH1 is up-regulated in CD44+/CD24− CSC population in both OE21 and OE33 cell lines according to a qPCR array of 84 genes related to cancer stemness. (A) Relative mRNA expression of CSC related genes in OE21 CD44+/CD24− CSC population, CD44+/CD24+ population and control (obtained from more differentiation form the cell lines derived xenograft tumors, [16]). Shown genes express >2 fold in CD44+/CD24− population compared to control. Control is set on 1. (B) Relative mRNA expression of CSC related genes in OE33 CD44+/CD24− CSC population, CD24+/CD44+ population and control. Shown genes express >2 fold in CD44+/CD24− population compared to control. (C) Validation of relative mRNA expression of PTCH1 in CD44+/CD24− CSC population, CD24+/CD44+ population and control in OE21 and OE33 by qPCR. PTCH1 was 3.8 fold up-regulated in CD44+/CD24− CSC population compared to control in OE21 (p = 0.04, N = 3) and 2.3 fold in OE33 (p = 0.04, N = 5). Error bars represent standard deviation.
Figure 4
Figure 4
Sonic Hedgehog, ligand of the PTCH1 receptor up-regulates sphere formation of both CD44+/CD24− and CD44+/CD24+ populations in OE21 and OE33 while vismodegib, a HH pathway inhibitor, deselects for the CD44+/CD24− population, decreases sphere formation and increases radiosensitivity. (A) Relative number of spheres formed in OE21 and OE33 after sorting for CD44+/CD24− and CD44+/CD24+ populations with the controls set on 1. Cells treated with SHH formed significantly more spheres in CD44+/CD24− and CD44+/CD24+ populations compared to their controls (DMSO-treated) (OE21: 1.97 (p = 0.009, N = 3) and 2.33 (p = 0.03, N = 3) fold respectively. OE33: 1.68 (p = 0.04, N = 3) and 2.18 (p = 0.04, N = 3) fold respectively. Spheres were counted after five days. (B) Percentage of CD44+/CD24− expression in OE21 and OE33 (unsorted populations) control (DMSO-treated) and vismodegib (5 nM) treated cells. Vismodegib treated cells showed significantly a deselection of the CD44+/CD24− phenotype in both cell lines compared to (DMSO-treated) controls (OE21 p = 0.02, OE33 p = 0.04, N = 4). (C) Amount of spheres formed in OE21 and OE33 control and vismodegib (5 nM) treated cells. Spheres were significantly lower in vismodegib treated cells compared to control (DMSO-treated) in OE21 (p = 0.008, N = 3) and OE33 (p = 0.03, N = 4). Survival of vismodegib-treated cells in response to (D) radiation and (E) Carboplatin. Error bars represent standard deviations, *p < 0.05, **p < 0.01.
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