HDAC4 degradation by combined TRAIL and valproic acid treatment induces apoptotic cell death of TRAIL-resistant head and neck cancer cells

Although TRAIL can directly induce cell death in some cancer cells, it appears that TRAIL resistance exists in many cancers. This study focuses on anti-cancer drugs for TRAIL-resistant head and neck cancer (HNC) to provide further progress toward effective cancer therapy. Results indicate in TRAIL-resistant HNC cells, that combined TRAIL and VPA treatment greatly reduced cell viability and therefore induced cell death, relative to treatment with TRAIL or VPA alone. A caspase-dependent signaling pathway was demonstrated, and combined treatment with TRAIL and VPA also significantly decreased the expression of HDAC4. When we pretreated cells with z-VAD followed by combined treatment with TRAIL and VPA, cell death was blocked with no reduction in expression of HDAC4. To confirm that cell death involved HDAC4 in HNC cells, we knocked down expression of HDAC4 with siRNA, followed by treatment with TRAIL and VPA. Results showed that loss of HDAC4 sensitized the TRAIL-resistant HNC cells to apoptotic cell death. Finally, we showed elevated expression of HDAC4 in HNC tissues compared to normal tissues obtained from the same patients. In conclusion, we suggest that combined VPA and TRAIL treatment may be a promising therapy for HNC via HDAC4 degradation.

HDACs are histone deacetylases that are counteracted by histone/lysine acetyltransferases 18 . There are class I HDACs (HDAC1, 2,3,8) and class II HDACs (HDAC4, 5,6,7,9,10) 19 . Class II HDACs are able to shuttle in and out of the nucleus in response to certain cellular signals 19 . Also, 14-3-3 protein is able to associate with HDAC4/5 or HDAC7 in the cytoplasm when phosphorylated, and these then enter the nucleus 18 . HDAC inhibitors have been known to induce cell cycle arrest, differentiation and apoptosis in vitro and in vivo 20 . Among HDAC inhibitors, valproic acid (VPA) has been reported to have anti-cancer activity due to transcriptional regulation of cell survival factors 21 .
In this study, we examined VPA to determine the sensitivity of HNC cells to TRAIL, which could result in apoptotic cell death, and in addition we identified the precise cell death mechanism. Our findings provide insights into the potential for combined treatment with TRAIL and VPA, which together induce apoptotic cell death via degradation of HDAC4. Our data suggest this approach has great potential as an anti-cancer strategy for head and neck cancers.

VPA induced cell death in TRAIL-resistant head and neck cancer cells. Previously, Sung et al.
reported several HDAC inhibitors induced the apoptotic cell death in TRAIL-resistant cells 22 . This sounded interesting and a good reference to us. Therefore, we wanted to apply it to this study and examine the head and neck cancer cells lines that are resistant to TRAIL. We selected VPA among HDAC inhibitors to investigate the combined effect of VPA and TRAIL on HNC cells (FaDu, HN3 and SNU899) and performed an MTT assay with cells following treatment with VPA (2.5, 5, 10, 20 mM) and/or TRAIL (0.5, 1 µg/ml) for 24 h.
As shown in Fig. 1A, FaDu and HN3 cells were resistant to TRAIL alone, but cell viability was decreased by combined treatment with VPA and TRAIL, however this was not the case for SNU899 cells. Moreover, cell death from combined VPA and TRAIL treatment was confirmed using Annexin-V/PI staining assay (Fig. 1B).
To examine the involvement of caspase activation in apoptotic cell death, we also pretreated cells with z-VAD (caspase 3, 6, 7 inhibitor) or caspase 9 inhibitor for 1 h followed by treatment with VPA and/or TRAIL for 24 h. We observed apoptotic cells by light microscopy (Fig. 1C) and confirmed that apoptotic cell death by combined treatment of VPA and TRAIL in HNC cells occurred in a caspase-dependent manner.
Cell surface receptor expression of DR4 and DR5 on HN3 cells. We tested the expression of functional death receptors (DR4 and DR5) to better understand how the mechanism of cell death through the combined treatment of TRAIL and VPA in HNC cell lines. According to the previous studies, it has been suggested that the upregulation of DR4 and DR5 by cancer drugs increased the TRAIL-induced cell death of cancer cells 23,24 . We assessed cell surface receptor expression of DR4 and DR5 using anti-DR4 and anti-DR5 antibodies on HN3 cells. Although it appeared that there was cell surface expression of DR4 and DR5 on HN3 cells ( Fig. 2A), TRAIL and VPA treatment did not increase their expression.

Combined TRAIL and VPA treatment induced the activation of apoptotic protein. Western blot-
ting was also performed using antibodies against DR4, DR5, Bax, XIAP, Bid, cleaved PARP, cleaved caspase 8, 7 and 3, HDAC 1, 2, 3, 4, 5 and 7 to see the activation of apoptotic molecules in HN3 cells treated with TRAIL and/ or VPA. The result showed that proapoptotic molecule BAX slightly increased and antiapoptotic molecules, XIAP and Bid decreased, and apoptotic cell death marker, cleaved caspase 3, cleaved PARP increased during treatment with the TRAIL and VPA combination, consistent with phenotype of the cell death (Fig. 2B). In this experiment, cleaved caspase 8 and cleaved caspase 7 do not seem to be related to the cell death induced by TRAIL/VPA combination, because those already showed the cleaved forms in cells treated with TRAIL alone, without phenotype of cell death. It was interesting enough to bring us a question that it is the combined treatment of TRAIL and VPA that induced the cell death, not TRAIL alone. Therefore we tried to identify the specific mechanism among HDAC molecules and observed the diminished expression of HDAC4 in the cells treated TRAIL and VPA not TRAIL alone. To support this finding, we also pretreated the cells with the related caspase inhibitors (z-VAD, caspase 3, 6 and 9 inhibitors), followed by combined treatment with TRAIL and VPA to find the relation between HDAC4 expression and cell death in HN3 cells (Fig. 2C). The results showed that HDAC4 was completely degraded by the combined TRAIL/VPA treatment and partially degraded with each caspase inhibitor.

Cell death induced by treatment with TRAIL/VPA affects expression of 14-3-3 proteins.
We also investigated another related signaling pathway in cell death by the combined treatment with TRAIL and VPA by doing Western blotting after the combined treatment for 3, 6, 12 and 24 h in HN3 cells. The critical roles of p38 or JNK signaling for activating the programmed cell death have been already shown previously 25 . In this experiment, we were able to observe the up-regulation of p-p38 at 6, 12, and 24 h during the cell death unlike the treatment with TRAIL alone. However, JNK signaling did not detected (data not shown), and the expression of Bcl-xL which blocks apoptotic cell death 26 was decreased. (Fig. 3A).
Previously, Wang et al. reported that HDAC4 was regulated by binding to 14-3-3 proteins 27 . To determine the involvement of 14-3-3 proteins in the degradation of HDAC4, we performed Western blotting after combined TRAIL and VPA treatment in HN3 cells. As shown in Fig. 3B, the expression of 14-3-3 proteins decreased during the process of TRAIL/VPA induced cell death (Fig. 3B). However, the binding of HDAC4 and 14-3-3 protein does not appear to be related to apoptosis (data not shown).

Knock-down of HDAC4 sensitized head and neck cancer cells to TRAIL-induced cell death.
It is obvious that highly expressed HDAC4 plays a critical role in TRAIL resistant cells. To confirm and support this fact, we knocked down the HDAC4 expression with HDAC4 siRNA in cells (Fig. 4). After transfection for 24 h, the transfected cells were treated with TRAIL alone or combined treatment of TRAIL and VPA. As a result, the cell death was observed in the TRAIL alone induced cells treated with HDAC4 siRNA, compared to control cells treated with siRNA-treated, which led us to a conclusion that HDAC degradation is important for cell death induced by TRAIL in TRAIL-resistant cells.
ScienTific RepoRts | (2018) 8:12520 | DOI:10.1038/s41598-018-31039-8 Expression of HDAC4 is upregulated in human HNC tissue. We performed more experiments with 10 cancer tissues from head and neck cancer patients. HDAC4 expression was tested by Western blotting with 10 cancer tissues and paired normal tissues. We observed more clear upregulation in HNC tissues compared to normal tissues (Fig. 5). Therefore, HDAC4 could be a good potential therapeutic target for HNC therapy.

Discussion
Head and neck cancers are highly heterogeneous and very difficult to cure. Therefore, there is an urgent need to develop additional therapeutic agents. TRAIL is a useful ligand to induce death in many cancer cells that overexpress the death receptors (DR4 and DR5) because it does not affect normal cells. Some cancers are resistant however. As a consequence there are many ongoing studies to try to overcome this resistance and discover pathways that achieve the therapeutic goals. Some studies have found an increase in the expression of DR4 or DR5 induced by combined treatment of multiple drugs and reported that TRAIL enhances the cell death of cancer    TRAIL-mediated apoptosis signaling is definitely a very crucial pathway involved with caspase 8 activity. In this case, as the Fig. 2B shows, the activity of cleaved caspase 8 is shown enough to see the involvement with only TRAIL itself. However, even though the combined treatment of TRAIL and VPA does show the activity of caspase 8, cell death doesn't occur. Therefore, we suspect that there should be something that controls the activity of effector caspases, which needs further study.
Interestingly, in this study, upregulation of DR4 and DR5 was not seen in the presence of TRAIL and VPA (Fig. 2B), which convinced us that another mechanism was likely involved in TRAIL/VPA induced cell death. Therefore, when we immunoblotted with anti-HDAC 1, 2, 3, 4, 5 and 7 antibodies, we focused more on a decrease in HDAC4 expression in the presence of both TRAIL and VPA, and not TRAIL or VPA alone. We also found that not only did z-VAD inhibit cell death induced by TRAIL/VPA, but also that it stopped HDAC4 degradation.
Consistent with our data, Zeng et al. showed that HDAC4 overexpression is important for the oncogenesis of esophageal carcinoma 30 , which supports the belief that the expression of HDAC4 in cancer cells is crucial to the carcinogenic potential.
Nishino et al. showed that 14-3-3 proteins regulate the nuclear import of class II a histone deacetylases 31 . To find out that the importance of the binding of HDAC with 14-3-3 proteins for the cell death occurred by the combined treatment, we tested for cell death using HDAC4(3SA) mutant which doesn't bind to 14-3-3 proteins. Surprisingly, the cell death still occurred with the combined treatment of VPA and TRAIL even with no binding of HDAC4 mutant with 14-3-3 proteins. Therefore, we came to a conclusion that the binding of HDAC4 and 14-3-3 has nothing to do with the cell death occurred by the combined treatment. (data not shown).
Our findings demonstrate that HDAC4 degradation plays an important role in cell death induced by the combined treatment of TRAIL and VPA in TRAIL-resistant cells. Therefore, we conclude that this drug combination may be an effective anticancer therapeutic agent to control head and neck cancer.