Mitochondrial dysfunction and epithelial to mesenchymal transition in head neck cancer cell lines

Abstract

Mitochondrial dysfunction promotes cancer aggressiveness, metastasis, and resistance to therapy. Similar traits are associated with epithelial mesenchymal transition (EMT). We questioned whether mitochondrial dysfunction induces EMT in head and neck cancer (HNC) cell lines. We induced mitochondrial dysfunction in four HNC cell lines with carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial electron transport chain uncoupling agent, and oligomycin, a mitochondrial ATP synthase inhibitor. Extracellular flux analyses and expression of the cystine/glutamate antiporter system xc (xCT) served to confirm mitochondrial dysfunction. Expression of the EMT-related transcription factor SNAI2, the mesenchymal marker vimentin and vimentin/cytokeratin double positivity served to detect EMT. In addition, holotomographic microscopy was used to search for morphological features of EMT. Extracellular flux analysis and xCT expression confirmed that FCCP/oligomycin induced mitochondrial dysfunction in all cell lines. Across the four cell lines, mitochondrial dysfunction resulted in an increase in relative SNAI2 expression from 8.5 ± 0.8 to 12.0 ± 1.1 (mean ± SEM; p = 0.007). This effect was predominantly caused by the CAL 27 cell line (increase from 2.2 ± 0.4 to 5.5 ± 1.0; p < 0.001). Similarly, only in CAL 27 cells vimentin expression increased from 2.2 ± 0.5 × 10^-3 to 33.2 ± 10.2 × 10^-3 (p = 0.002) and vimentin/cytokeratin double positive cells increased from 34.7 ± 5.1 to 67.5 ± 9.8% (p = 0.003), while the other 3 cell lines did not respond with EMT (all p > 0.1). Across all cell lines, FCCP/oligomycin had no effect on EMT characteristics in holotomographic microscopy. Mitochondrial dysfunction induced EMT in 1 of 4 HNC cell lines. Given the heterogeneity of HNC, mitochondrial dysfunction may be sporadically induced by EMT, but EMT does not explain the tumor promoting effects of mitochondrial dysfunction in general.

Figure 1

Holotomographic microscopy scoring example of EMT characteristics (SCC25). (A) Score 0 for fibroblast-like-morphology (FM). (B) Score 1 for FM. (C) Score 2 for FM and (D) score 3 for FM. (E) Score 0 for cell individualization. (F) Score 1 for cell individualization. (G) Score 2 for cell individualization and (H) score 3 for cell individualization. (I) Score 0 for surface adhesion, (J) Score 1 for surface adhesion. (K) Score 2 for surface adhesion and (L) Score 3 for surface adhesion. Live cell imager (Nanolive, Switzerland); 20 μm.

Figure 2

Relative SNAI2 (A), vimentin (VIM; B) expression, and percentage of vimentin/cytokeratin double positive cells (C) in response to FCCP/oligomycin (EMM: Estimated marginal mean; Bars: SEM; *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3

Holotomographic microscopy of HN cells (A) in response to FCCP/oligomycin addition (A1 + A2) and SCC25 cells (B) in response to TGF-ß1 (B1 + B2). Bars: 20 μm.

Figure 4

OCR/ECAR ratio (A) and xCT expression (B) of 4 HNSCC cell lines in response to FCCP/oligomycin (EMM: Estimated marginal mean; bars: SEM; *p < 0.05, **p < 0.01, ***p < 0.001).