Combination of ACY-241 and JQ1 Synergistically Suppresses Metastasis of HNSCC via Regulation of MMP-2 and MMP-9

Abstract

Overexpression of histone deacetylase 6 (HDAC6) and bromodomain-containing protein 4 (BRD4) is related to aggressiveness of head and neck squamous carcinoma (HNSCC). Based on studies that HDAC6 and BRD4 are potential therapeutic targets of HNSCC, we hypothesized that the combination treatment of BET inhibitor JQ1 and HDAC6-selective inhibitor ACY-241 could exhibit synergistic anticancer effects in human papillomavirus (HPV)-positive and HPV-negative HNSCC cells. In this study, HNSCC cell growth and viability were measured by CCK-8 assay, apoptosis was analyzed by flow cytometry, and metastasis was studied by wound healing and transwell assays. Furthermore, immunoblotting is conducted to investigate proteins that modulate apoptosis or metastasis. Here, we report that the combination of ACY-241 and JQ1 shows synergistic cell growth inhibition, viability reduction, and apoptosis induction in HNSCC cells through inactivation of AKT and NF-κB signaling. Importantly, we demonstrate that combined treatment of ACY-241 and JQ1 synergistically suppresses TNF-α-induced migration and invasion via dysregulating matrix metalloproteinase (MMP)-2, MMP-9, and MT1-MMP. Overall, the combination of ACY-241 and JQ1 significantly suppresses proliferation and metastasis in HPV-positive and HPV-negative HNSCC. Collectively, these findings suggest that the co-inhibition of BET and HDAC6 can be a new therapeutic strategy in HNSCC.

Keywords: ACY-241; HDAC6; HNSCC; HPV; JQ1; metastasis.

Figure 1

Single treatments of ACY-241 and JQ1 time- and dose-dependently suppress cell growth and reduce cell viability in both human papillomavirus (HPV)-positive and HPV-negative head and neck squamous carcinoma (HNSCC) cells. (A,E) 2A3 cells and (B,F) FaDu cells were treated with 0.1% DMSO or ACY-241 at indicated concentrations for 24–72 h. (C,G) 2A3 cells and (D,H) FaDu cells were treated with 0.1% DMSO or JQ1 at indicated concentrations for 24–72 h. CCK-8 assay was performed to measure cell growth (A–D) and viability (E–H). Values represent mean ± SD from three independent experiments (n = 3). * p < 0.05, ** p < 0.01, or *** p < 0.001 vs. DMSO control.

Figure 2

Combination treatment of ACY-241 and JQ1 synergistically decreases HNSCC cell viability. (A,B) ACY-241 and JQ1 were treated alone or in combination at a 2:1 ratio to 2A3 cells and FaDu cells. Cell viability was measured by CCK-8 assay (48 h). Synergism of ACY-241 and JQ1 was determined by combination index (CI) using Chou–Talalay method. (C,D) Inhibitory enzymatic effects of ACY-241 and JQ1 were confirmed by immunoblot analysis of acetyl α-tubulin and c-Myc, respectively. α-tubulin and GAPDH were used as loading controls. Protein levels were quantified relative to the loading control. Total protein was extracted after 24 h of ACY-241 (4 μM) or JQ1 (2 μM) treatment alone or in combination. Values represent mean ± SD (n = 3). * p < 0.05, ** p < 0.01, or *** p < 0.001 vs. DMSO control, ^$ p < 0.05, ^$$ p < 0.01, or ^$$$ p < 0.001 vs. ACY-241-treated group, ^# p < 0.05, ^## p < 0.01, or ^### p < 0.001 vs. JQ1-treated group. ns = not significant.

Figure 3

Combination treatment of ACY-241 and JQ1 synergistically induces apoptosis in HNSCC. (A,B) Immunoblot analysis of pro-apoptotic proteins (PARP, Cas-3) and anti-apoptotic proteins (XIAP, Bcl-xL) in 2A3 and FaDu cells. α-tubulin and GAPDH were used as loading controls. Protein levels were quantified relative to the loading control. Total protein was extracted after 24 h of ACY-241 (4 μM) or JQ1 (2 μM) treatment alone or in combination. (C,D) Flow cytometry analysis of 2A3 and FaDu cells. Cells were treated with 0.2% DMSO, ACY-241 (4 μM), or JQ1 (2 μM) alone or in combination for 72 h. 2A3 and FaDu cells were stained with annexin V and PI for 15 min. Values represent mean ± SD (n = 3). * p < 0.05, ** p < 0.01, or *** p < 0.001 vs. DMSO control, ^$ p < 0.05, ^$$ p < 0.01, or ^$$$ p < 0.001 vs. ACY-241-treated group, ^# p < 0.05, ^## p < 0.01, or ^### p < 0.001 vs. JQ1-treated group.

Figure 4

Combination treatment of ACY-241 and JQ1 synergistically downregulates MMP-2 and MMP-9 via inhibition of TNF-α/AKT/-NF-κB cascade in HNSCC. (A,B) Immunoblot analysis of Snail and MMP proteins (MMP-2, MMP-9, MT1-MMP) in 2A3 and FaDu cells. (C,D) Immunoblot analysis of EMT signaling factors (TNF-α, AKT, NF-κB) in 2A3 and FaDu cells. GAPDH was used as a loading control. Protein levels were quantified relative to the loading control. Total protein was extracted after 24 h of ACY-241 (4 μM) or JQ1 (2 μM) treatment alone or in combination. Values represent mean ± SD (n = 3). * p < 0.05, ** p < 0.01, or *** p < 0.001 vs. DMSO control, ^$ p < 0.05, ^$$ p < 0.01, or ^$$$ p < 0.001 vs. ACY-241-treated group, ^# p < 0.05 or ^## p < 0.01 vs. JQ1-treated group.

Figure 5

Combination treatment of ACY-241 and JQ1 synergistically inhibit HNSCC cell invasion and migration. (A,B) Wound healing assay of 2A3 and FaDu cells. Cells were seeded in 6-well plates and then scratched after cell adhesion. Artificial lines were drawn on microscopic images to visualize the scratched area. Percentage of wound closure was determined by the difference between wound areas in 0 h and 48 h. The wound was photographed at 50× magnification. Scale bar = 50 μm. (C) Transwell migration assay and (D) transwell invasion assay of 2A3 and FaDu cells. The cells were photographed at 200× magnification. Scale bar = 100 μm. Matrigel (0.4 mg/mL) was coated on the inner inserts of transwell plate for 1 h during invasion assay. Cells were treated with 0.2% DMSO, ACY-241 (4 μM), and JQ1 (2 μM) alone or in combination for 48 h. Data were normalized by cell viability to represent migration and invasion of viable cells. Values represent mean ± SD (n = 3). * p < 0.05, or *** p < 0.001 vs. DMSO control, ^$$$ p < 0.001 vs. ACY-241-treated group, ^# p < 0.05, ^## p < 0.01 or ^### p < 0.001 vs. JQ1-treated group.

Figure 6

Schematic diagram of post-translational modulations underlying synergistic anticancer effects induced by ACY-241 and JQ1 combination treatment in HNSCC. ACY-241 and JQ1 dysregulate MMP-2 and MMP-9 expression and secretion by suppressing TNF-α, an inducer of active AKT and NF-κB. Migration, invasion, and metastasis of HNSCC cells are synergistically inhibited by ACY-241 and JQ1 treatments.