Inhibition of HDAC6 Protein Enhances Bortezomib-induced Apoptosis in Head and Neck Squamous Cell Carcinoma (HNSCC) by Reducing Autophagy

Insoon Chang,Cun-Yu Wang

doi:10.1074/jbc.m116.717793

Abstract

Chemoresistance is a major barrier to effective chemotherapy of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Recently, autophagy, a highly conservative intracellular recycling system, has shown to be associated with chemoresistance in cancer cells. However, little is known about how autophagy plays a role in the development of chemoresistance in HNSCC and how autophagy is initiated when HNSCC cells undergo cytotoxic stress. Here, we report that autophagy was activated when HNSCC cells are treated with the proteasome inhibitor bortezomib, proposed as an alternative chemotherapeutic agent for both primary and cisplatin-resistant HNSCC cells. Ablation of histone deacetylase 6 (HDAC6) expression and its activity in HNSCC cells significantly inhibited autophagy induction by altering the phosphorylation status of mammalian target of rapamycin and enhanced the bortezomib cytotoxicity. Similarly, a combination regimen of bortezomib and the histone deacetylase inhibitor trichostatin A abolished HDAC6 activity and decreased autophagy induction while significantly enhancing bortezomib-induced apoptosis in HNSCC cells. These data uncover a novel molecular mechanism indicating that HDAC6 may serve as a critical causal link between autophagy, apoptosis, and the cell survival response in HNSCC. A combination regimen resulting in regression of autophagy improves chemotherapeutic efficacy, thereby providing a new strategy to overcome chemoresistance and to improve the treatment and survival of HNSCC patients.

Highlights

Major cancer [3, 4]
Btz Induces Both Autophagy and Apoptosis in head and neck squamous cell carcinoma (HNSCC) Cells and Inhibition of Autophagy Enhances the Apoptosis—In our previous work, we showed that Btz induced apoptosis in HNSCC cell lines, including SCC1 and SCC23, which could be synergistically enhanced by TSA [7, 8, 11]
Western blotting showed that autophagy protein 5 (ATG5) knockdown down-regulated autophagy activation but enhanced caspase-9 activation induced by Btz (Fig. 1E)

Summary

Results

Btz Induces Both Autophagy and Apoptosis in HNSCC Cells and Inhibition of Autophagy Enhances the Apoptosis—In our previous work, we showed that Btz induced apoptosis in HNSCC cell lines, including SCC1 and SCC23, which could be synergistically enhanced by TSA [7, 8, 11]. Western blot analysis revealed that both LC3-I and LC3-II expression increased in a time-dependent manner in SCC1 cells following Btz treatment, indicating activation of autophagy (Fig. 1A). Treatment with Btz showed a significant increase in apoptosis in ATG5-knockdown (ATG5KD) SCC1 cells compared with the control cells (Fig. 1, C and D). We found that TSA inhibited autophagy activation induced by Btz in SCC23 cells as determined by GFP puncta formation assays (Fig. 3E), flow cytometry (Fig. 3F), and Western blotting (Fig. 3G). To determine whether HDAC6 played a role in Btz-induced aggresome formation and autophagy induction in SCC cells, we stably knocked down HDAC6 using shRNA (HDAC6KD) in SCC1 cells (Fig. 4A).

80 ATG5KD

Discussion

F DMSO 110

Experimental Procedures