Ricolinostat (ACY-1215) suppresses proliferation and promotes apoptosis in esophageal squamous cell carcinoma via miR-30d/PI3K/AKT/mTOR and ERK pathways

Abstract

Ricolinostat (ACY-1215), a first-in-class selective HDAC6 inhibitor, exhibits antitumor effects alone or in combination with other drugs in various cancers. However, its efficacy in esophageal cancer remains unclear. In this study, we found that the high expression of HDAC6 was associated with poor prognosis in esophageal squamous cell carcinoma (ESCC) tissues. Then, we identified that ACY-1215 significantly inhibited cellular proliferation in ESCC, and caused G2/M phase arrest and apoptosis. We further demonstrated that ACY-1215 treatment reduced the expression of PI3K, P-AKT, P-mTOR, and P-ERK1/2 and increased that of Ac-H3K9 and Ac-H4K8. In addition, using miRNA microarray and bioinformatics analysis, we detected that ACY-1215 promoted miR-30d expression, and PI3K regulatory subunit 2 (PIK3R2) was a direct target of miR-30d. Anti-miR-30d partially rescued the G2/M phase arrest and apoptosis caused by ACY-1215 treatment. The reductions in PI3K, P-AKT, and P-mTOR expression were also partially reversed by miR-30d inhibitor. Furthermore, the effects of ACY-1215 inhibited ESCC proliferation were validated in a mouse xenograft model in vivo. In conclusion, our study showed that ACY-1215 suppressed proliferation and promoted apoptosis in ESCC via miR-30d/PI3K/AKT/mTOR and ERK pathways and that ACY-1215 may be a promising antitumor agent in ESCC.

Fig. 1. The high expression of HDAC6 was associated with poor prognosis in ESCC specimens, and ACY-1215-inhibited tumor cell proliferation.

a The optimal cutoff values for HDAC6-related mRNA levels were determined and Kaplan–Meier survival curves for patients with ESCC by HDAC6-relatived mRNA levels. b ACY-1215 treatment significantly inhibited cellular proliferation that was dose- and time-dependent manner in EC109 (IC50: 46 μM), KYSE150 (IC50: 57 μM), TE-1 (IC50: 45 μM) and TE-13 (IC50: 37 μM) cells. c The relative levels of HDAC6 expression were higher in all ESCC cell lines than that of the HUVEC cell. d ACY-1215 inhibited cellular proliferation in HUVEC is significantly less than EC109 and TE-1 cells (24 h). The results are shown as the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 2. ACY-1215 treatment induced G2/M-phase cell cycle arrest.

a, c ESCC cell lines were treated with ACY-1215 for 48 h. Cells were then stained with propidium iodide, and analyzed by LSR II flow cytometer. ACY-1215 caused a significant increase in the G2/M phase and a decrease in the G1 phase, occurred in a dose-dependent manner. b, d Western blot analysis of G2/M phase cell cycle regulatory-proteins for 48 h

Fig. 3. ACY-1215 treatment induced tumor cell apoptosis.

a, c ESCC cell lines were treated with ACY-1215 for 48 h. ACY-1215 caused a significant increase the proportion of apoptosis cells in a dose-dependent manner. b, d Western blot analysis of apoptosis regulatory-proteins for 48 h

Fig. 4. ACY-1215 inhibited PI3K/AKT/mTOR and ERK signaling and increased intranuclear histone expression.

a ACY-1215 treatment inhibited PI3K/AKT/mTOR and ERK signaling protein levels in ESCC EC109 and TE-1 cell lines for 48 h. b GSK690693 (10 μM) combined with ACY-1215 (50 μM) treatment further enhanced the ACY-1215-inhibited cell proliferation (48 h). c GSK690693 (10 μM) increased P-AKT due to blocking a negative feedback loop downstream of AKT (PRAS40, Rag C and P-mTOR) for 48 h. d ACY-1215 treatment increased intranuclear histones (Ac-H3K9 and Ac-H4K8) levels in ESCC cell lines for 48 h

Fig. 5. ACY-1215 promoted miR-30d expression, and miR-30d directly downregulated PI3K.

a ACY-1215 treatment significant increased miR-30d expression in EC109 and TE-1 cells for 48 h (50 μM). b ACY-1215 treatment significant reduced PIK3R2 expression in EC109 and TE-1 cells for 48 h (50 μM). c, d Cells were treated with Lipofectamine 2000, 50 μM ACY-1215, and/or 50 nM miR-30d inhibitor for 48 h. The quantitative real-time PCR analysis demonstrated that PIK3R2 expression significantly increased after cells were transfected with miR-30d inhibitor, particularly in the ACY-1215 treatment group. The results are shown as the mean ± SD. **p < 0.01; ***p < 0.001

Fig. 6. Anti-miR-30d partially restored the G2/M arrest and apoptosis and AKT signaling caused by ACY-1215 treatment.

a, c The ACY-1215 (50 μM) induced G2/M phase cell cycle arrest was partially rescued by anti-miR-30d (50 nM). Cells were treated with Lipofectamine 2000, 50 μM ACY-1215, and/or 50 nM miR-30d inhibitor for 48 h. b, d The ACY-1215 (50 μM) induced apoptosis was partially rescued by anti-miR-30d (50 nM). Cells were treated with Lipofectamine 2000, 50 μM ACY-1215, and/or 50 nM miR-30d inhibitor for 48 h. The results are shown as the mean ± SD. **p < 0.01; ***p < 0.001

Fig. 7. Cells were treated with Lipofectamine 2000, 50 μM ACY-1215, and/or 50 nM miR-30d inhibitor for 48 h.

Western blot analysis demonstrated that the ACY-1215 induced changes in the expression of cell cycle-regulatory and apoptosis-regulatory proteins and AKT signaling proteins were also partially reversed by anti-miR-30d

Fig. 8. ACY-1215 treatment led to significant tumor growth slowed in vivo.

a, b EC109 cells were injected subcutaneously into the right side of each BALB/c male nude mice. After three cycle of therapy, animals were sacrificed and tumors were excised. c Tumor diameters were measured after therapy 3 times every week, and tumor volumes were calculated. Values are mean ± SD. *p < 0.05