Hypoxia Mediates Differential Response to Anti-EGFR Therapy in HNSCC Cells

Abstract

Despite advances in the head and neck squamous cell carcinoma (HNSCC) treatment modalities, drug resistance and cancer recurrence are often reported. Hypoxia signaling through hypoxia-inducible factor 1 (HIF-1) promotes angiogenesis and metastasis by inducing epithelial-mesenchymal-transition (EMT). The aim of this study was to evaluate the impact of hypoxia on response to therapy as well as EMT and expression of stem cell markers in HNSCC cells. Five HNSCC cell lines (UT-SCC-2, UT-SCC-14, LK0412, LK0827, and LK0923) were selected for this study. The treatment sensitivity for radiation, cisplatin, cetuximab, and dasatinib was assessed by crystal violet assay. Gene expression of EMT and cancer stem cell (CSC) markers as well as protein level of EGFR signaling molecules were analyzed by qPCR and western blotting, respectively. Unlike UT-SCC-14 and LK0827, the LK0412 cell line became significantly more sensitive to cetuximab in hypoxic conditions. This cetuximab sensitivity was efficiently reversed after suppression of HIF-1α with siRNA. Additionally, hypoxia-induced EMT and expression of stem cell markers in HNSCC cells was partially revoked by treatment with cetuximab or knockdown of HIF-1α. In summary, our study shows that hypoxia might have a positive influence on the anti-EGFR therapy effectiveness in HNSCC. However, due to heterogeneity of HNSCC lesions, targeting HIF-1α may not be sufficient to mediate such a response. Further studies identifying a trait of hypoxia-specific response to cetuximab in HNSCC are advisable.

Keywords: HIF-1α; cancer stem cells (CSC); cetuximab; cisplatin; epithelial-mesenchymal transition (EMT); head and neck tumors; hypoxia; radiotherapy.

Figure 1

Hypoxia-induced treatment response in Head and Neck Squamous Cell Carcinoma (HNSCC) cell lines. Cell lines were cultured in normoxia and hypoxia. (A) After 24 h, they were treated with various concentrations of cetuximab (cet; nM), cisplatin (cis; µg/mL), dasatinib (das; nM), and radiation (2, 4, 6 Gy). After 9 days the cytotoxic/cytostatic effect on cell proliferation was determined by crystal violet assay. Cell proliferation is presented as the percentage of the untreated controls (mean values ± SD; n = 3, triplicates). For statistical analysis, one-way ANOVA with post-hoc Bonferroni analysis was used (* p < 0.05; ** p < 0.01; # p < 0.001); (B) Western blot analysis of hypoxia-inducible factor (HIF)-1α expression in normal oral human keratinocytes (NOHK) as well as UT-SCC-2, UT-SCC-14, LK0412, LK0827, and LK0923 HNSCC cells. Hypoxic cells were exposed to cetuximab (60 nM) for 3 days prior to harvesting for Western blotting; β-actin was used as the loading control. Abbreviations: N, normoxia; H, hypoxia; H + Cx, hypoxia in the presence of cetuximab; Cx, cetuximab.

Figure 2

Hypoxia-induced epithelial-mesenchymal transition (EMT) and expression of stem cell markers in HNSCC. RT-qPCR was performed to analyze mRNA expression levels of EMT (A) and stem cell (B) markers in HNSCC cells following exposure to normoxic and hypoxic conditions for 7 days in the presence or absence of cetuximab (60 nM). The relative amount of analyzed genes is calculated using the 2^−ΔΔCt method and amplification of GAPDH and β-actin were used as an internal standard. Data were normalized to cells cultured in normoxic conditions in each column graph (mean values ± SD; n = 3). * p < 0.05 versus N (normoxia) and ** p < 0.05 versus H (hypoxia) according to Student’s t-test.

Figure 3

Effect of HIF-1α downregulation on drug response in HNSCC. The UT-SCC-14 and LK0412 cell lines were transiently transfected with either non-targeting siRNA (control siRNA) or HIF-1α-specific siRNA. The protein expression of HIF-1α was assessed by western blotting (A) and the mRNA expression level of HIF-1α was assessed by RT-qPCR (B) after 72 h of incubation in hypoxic conditions. (C,D) Cells were treated with various concentrations of cetuximab (cet; nM) and cisplatin (cis; µg/mL) 24 h post-transfection with either non-targeting siRNA (control siRNA) or HIF-1α-specific siRNA followed by exposure to hypoxic conditions. After 9 days the cytotoxic/cytostatic effect on cell proliferation was determined by crystal violet assay. Cell proliferation is presented as the percentage of the untreated controls (mean values ± SD; n = 3, triplicates). For statistical analysis, one-way ANOVA with post-hoc Bonferroni analysis was used (* p < 0.05).

Figure 4

Effect of HIF-1α downregulation on EMT profile and expression of stem cell markers in HNSCC. The UT-SCC-14 and LK0412 cells were transiently transfected with either non-targeting siRNA or HIF-1α-specific siRNA and maintained under hypoxia for 72 h. The mRNA expression levels of (A) EMT markers and (B) stem cell markers in HNSCC cells cultured under hypoxia were analyzed by RT-qPCR. The relative amount of analyzed genes is calculated using the 2^–ΔΔCt method and amplification of GAPDH and β-actin were used as an internal control. Data were normalized to cells cultured in hypoxic conditions and transfected with control, non-targeting siRNA in each column graph (mean values ± SEM; n = 3). * p < 0.05 according to Student’s t-test.

Figure 5

Effect of hypoxia on EGFR downstream signaling in HNSCC cell lines. Western blot analysis of the EGFR signaling pathway in LK042 and UT-SCC-14 HNSCC cells treated with 60 nM of cetuximab for 3 days; GAPDH was used as the loading control. Abbreviations: N, normoxia; N + Cx, normoxia in the presence of cetuximab; H, hypoxia; H + Cx, hypoxia in the presence of cetuximab; Cx, cetuximab.