Involvement of c-Fos in the Promotion of Cancer Stem-like Cell Properties in Head and Neck Squamous Cell Carcinoma

Abstract

Purpose: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Although improvements in surgical techniques, chemotherapy and radiation delivery, and supportive care have improved quality of life for patients with HNSCC, regional and distant recurrence remain common. Recent evidence suggests that cancer stem-like cells (CSC) play a significant role in recurrence and chemoresistance. We previously observed that c-Fos was highly upregulated in the HNSCC sphere-forming cells. Consequences of c-Fos upregulation for the biology of HNSCC-CSCs are poorly understood. In this study, we investigated the role of c-Fos in renewal of stemness of HNSCC and tumor growth.Experimental Design and Results: We generated stable HNSCC cell lines ectopically expressing the c-Fos gene. Exogenous expression of c-Fos in nontumorigenic MDA1386Tu cells makes these cells tumorigenic in nude mice. Furthermore, subcutaneous transplantation of c-Fos-overexpressing Cal27 cells (tumorigenic) into immunocompromised mice enhanced tumor growth as compared with parental cells. Mechanistic investigations demonstrated that c-Fos overexpression enhanced the epithelial-mesenchymal transition (EMT) state and expression of CSC markers (Nanog, c-Myc, Sox2, and Notch1). Ectopic expression of c-Fos in HNSCC cells also displays increased sphere formation. We further observed that overexpression of c-Fos increased the expression of pERK and cyclin D1 in HNSCC cells.Conclusions: Together, our results strongly suggest a novel role of c-Fos as a regulator of EMT and cancer stem cell reprogramming in HNSCC cells, which may hold potential as a CSC-directed therapeutic approach to improve HNSCC treatment. Clin Cancer Res; 23(12); 3120-8. ©2016 AACR.

Figure 1

Exogenous expression of c-Fos augments tumor growth in xenograft mouse models. (A) Cal27-control, Cal27-c-Fos cells, MDA1386Tu-control and MDA1386Tu-c-Fos cells lysates were subjected to Western blot analysis using c-Fos antibody. The blots were reprobed with an antibody to actin for comparison of protein loading in each lane. Densitometric analysis of c-Fos were done by using Image J software and shown on the right. (B) Cal27 and (C) MDA1386Tu c-Fos overexpressing and their respective control cells were injected into the flanks of each nude mouse. Volume of tumor growth was measured as indicated times and presented as a mean ±SD. Small bar indicates standard error (*, p < 0.05). (D) Hematoxylin and eosin (H & E) stain image displayed spindle shaped cancer cells from MDA1386Tu-c-Fos overexpressing cells. Images are taken at 40X.

Figure 2

Increased expression of vimentin from c-Fos overexpressing MDA1386Tu cells. (A) Lysates from Cal27, MDA1386Tu control and c-Fos expressing cells and tumors are subjected to Western blot analysis for vimentin and cytokeratin expression using specific antibodies. Blots are reprobed with an antibody to actin for comparison of protein loading in each lane. (B) Immunohistochemistry images showing expression of vimentin and cytokeratin from MDA1386Tu-c-Fos cells implanted tumors. Images are taken at 40x.

Figure 3

Exogenous expression of c-Fos alters cell proliferation and its related proteins. (A) Cal27-control and Cal27-c-Fos cells, and (B) MDA1386Tu-control and MDA1386Tu-c-Fos cells were plated in 35 mm plates in triplicates, and live cells were counted at indicated time points by using Trypan blue exclusion method. Small bar indicates standard error. (C) HNSCC cells lysates were subjected to Western blot analysis for pERK1/2 and ERK1/2 expression using specific antibodies. The blots were reprobed with an antibody to actin for comparison of protein loading in each lane. Densitometric analysis of pERK1/2 were done by using Image J software and shown on the right. (D) HNSCC cells lysates were subjected to Western blot analysis using antibody to cyclin D1. The blots were reprobed with an antibody to actin for comparison of protein loading in each lane. Densitometric analysis of c-Fos and cyclin D1 were done by using Image J software and shown on the right.

Figure 4

Exogenous expression of c-Fos enhances sphere formation. (A) Equal number of Cal27-control, Cal27-c-Fos cells and (B) MDA1386Tu-control and MDA1386Tu-c-Fos cells were seeded in ultra-low attachment plates and incubated for 10 days. The number of spheres (>75 mm) were counted. The results are presented as means of three different experiments with standard deviations (*p < 0.05, **p < 0.01). Representative images of spheres are shown on the right.

Figure 5

Exogenous expression of c-Fos enhances the expression stemness related markers. Cal27-control, Cal27-c-Fos, MDA1386Tu-control and MDA1386Tu-c-Fos cells lysates were subjected to Western blot analysis for (A) Notch1 and Sox2, (B) Nanog and c-Myc, and (C) c-Met expression using specific antibodies. The blots were reprobed with an antibody to actin for comparison of protein loading in each lane. Densitometric analysis were done by using Image J software and shown on the right.

Figure 6

Exogenous expression of c-Fos enhances the migration of cells and VEGF promoter activity. (A) MDA1386Tu control and MDA1386Tu-c-Fos cells were seeded at confluency. Representative images of wound healing (0 and 24 hours after scratch) in MDA1386Tu cells expressing control plasmid DNA or c-Fos are shown. Control MDA1386Tu and MDA1386Tu-c-Fos cells were transfected with 2.6 Kb (B) or 0.35 Kb (C) VEGF promoter sequences, and luciferase activities were measured. The results are presented as means of three different experiments with standard deviations (**p < 0.01).