Radiation Sensitization of Basal Cell and Head and Neck Squamous Cell Carcinoma by the Hedgehog Pathway Inhibitor Vismodegib

Abstract

Vismodegib, an inhibitor of the Hedgehog signaling pathway, is an approved drug for monotherapy in locally advanced or metastatic basal cell carcinoma (BCC). Data on combined modality treatment by vismodegib and radiation therapy, however, are rare. In the present study, we examined the radiation sensitizing effects of vismodegib by analyzing viability, cell cycle distribution, cell death, DNA damage repair and clonogenic survival in three-dimensional cultures of a BCC and a head and neck squamous cell carcinoma (HNSCC) cell line. We found that vismodegib decreases expression of the Hedgehog target genes glioma-associated oncogene homologue (GLI1) and the inhibitor of apoptosis protein (IAP) Survivin in a cell line- and irradiation-dependent manner, most pronounced in squamous cell carcinoma (SCC) cells. Furthermore, vismodegib significantly reduced proliferation in both cell lines, while additional irradiation only slightly further impacted on viability. Analyses of cell cycle distribution and cell death induction indicated a G1 arrest in BCC and a G2 arrest in HNSCC cells and an increased fraction of cells in SubG1 phase following combined treatment. Moreover, a significant rise in the number of phosphorylated histone-2AX/p53-binding protein 1 (γH2AX/53BP1) foci in vismodegib- and radiation-treated cells was associated with a significant radiosensitization of both cell lines. In summary, these findings indicate that inhibition of the Hedgehog signaling pathway may increase cellular radiation response in BCC and HNSCC cells.

Keywords: basal cell carcinoma; head and neck squamous cell carcinoma; hedgehog signaling pathway; radiotherapy resistance; vismodegib (GDC-0449).

Figure 1

Vismodegib reduces the number of viable BCC-1 and SCC-25 cells. Relative quantification of cellular metabolic activity (i.e., number of viable cells) was performed for cells treated with vismodegib compared to dimethyl sulfoxide (DMSO) controls using a CellTiter 96^® Aqueous One Solution Cell Proliferation (MTS) Assay. (A) Time schedule of vismodegib treatment and measurement of colorimetric cell proliferation assay. BCC-1 or SCC-25 cells were plated 24 h before treatment with vismodegib for 3 to 96 h before analysis. Metabolic activity is expressed as relative absorption at 490 nm. This assay was carried out in five independent experiments (each in quadruplicate) for BCC-1 (B) and SCC-25 (C) cells. The metabolic activity of controls was set to 1.0 (i.e., reference value) as indicated. Statistical significance was assessed by t-test; * p < 0.05, ** p < 0.01 (vismodegib- versus DMSO-treated cells). BCC, basal cell carcinoma; Rel., relative; SCC, squamous cell carcinoma; Vism., vismodegib.

Figure 2

Vismodegib decreases hedgehog (Hh) target gene glioma-associated oncogene homologue 1 (GLI1) and Survivin expression. (A) Time schedule of vismodegib application and RNA/protein extraction for analysis. BCC-1 or SCC-25 cells were plated 24 h before treatment with 10 or 40 µM vismodegib or with DMSO as control for 24 h or 48 h before analysis. (B) mRNA expression for GLI1 and Survivin (C) relative to DMSO-treated controls. n = 2 (in duplicate); * p < 0.05, ** p < 0.01 (vismodegib- versus DMSO-treated cells, t-test). (D) Representative Western blots from at least two independent experiments, including detection of p53 expression in the cell lines. β-actin served as loading control. Rel., relative; Vism., vismodegib.

Figure 3

Vismodegib and irradiation modulate cell viability, cell cycle distribution and SubG1 cell fraction content. BCC-1 and SCC-25 cells were pretreated for 24 h with indicated concentrations of vismodegib or DMSO as control before a 4 Gy irradiation (A). At 24 h after irradiation, proliferation/viability was measured with a CellTiter 96^® Aqueous One Solution Cell Proliferation (MTS) Assay (B). Cell cycle distribution (C) and SubG1 cell fraction (D) were analyzed after propidium iodide staining by flow cytometric quantification. Caspase 3 and PARP expression/cleavage was detected by Western blotting (n = 2) with β-actin as loading control (E). Data given in (B–D) are shown as means + SD from four independent experiments with quadruplicates (MTS assay, (A)) or duplicates (flow cytometry (B,C)). Differences were considered as statistically significant when * p < 0.05 or highly significant when ** p < 0.01; vismodegib- versus DMSO-treated cells (t-test). Significant differences between irradiated and non-irradiated cells are indicated as follows: ^# p < 0.05, ^## p < 0.01 (t-test). Gy, Gray; PARP, poly ((adenosine diphosphate)ADP-ribose) polymerase; Rel., relative; Vism., vismodegib.

Figure 4

Vismodegib increases radiation-induced γH2AX/53BP1 nuclear foci. BCC-1 or SCC-25 cells were treated for 24 h with 10 or 40 µM vismodegib or with DMSO as control followed by irradiation with 4 Gy. (A) Representative images of phosphorylated gamma histone-2AX (γH2AX)/p53-binding protein 1 (53BP1) foci from non-irradiated and 4 Gy-irradiated DMSO control versus 40 µM vismodegib-treated cells are shown. Nuclei were counterstained with DAPI. Scale bar, 10 µm. (B) Quantification of persistent γH2AX/53BP1 foci in vismodegib treated and irradiated BCC-1 (left panel) and SCC-25 (right panel) cells. Bars represent means + SD from three independent experiments performed in duplicate. ** p < 0.01 vismodegib- versus DMSO-treated cells and ^# p < 0.05, ^## p < 0.01 4 Gy versus non-irradiated cells (t-test). Gy, Gray; Vism., vismodegib.

Figure 5

Vismodegib sensitizes basal and squamous cell carcinoma cells to ionizing radiation in a 3D clonogenic assay. BCC-1 and SCC-25 cells were plated as single cells in a three-dimensional (3D) laminin-rich extracellular matrix and were treated with vismodegib 24 h before irradiation with 0 to 8 Gy, single doses. (A) 7 days after plating, representative images of 3D grown colonies were acquired. Scale bar, 100 µm. (B) Colonies were counted microscopically from three independent experiments performed in triplicate. Basal plating efficiencies of cells treated with 5 to 40 µM vismodegib, are shown relative to DMSO-treated control cells. (C) Surviving fractions and radiation survival curves were calculated as described in the Materials and Methods section. Values represent means ± SD (n = 3). * p < 0.05, ** p < 0.01; vismodegib-treated cells versus DMSO control (t-test). Gy, Gray; Vism., vismodegib.