# Inhibition of _Tp53_ Mutant Oral Cancer by Reactivating p53
## 4 Discussion  
Loss of function mutation in _TP53_ is frequently found in oral cancers [3; 4]. As p53 is a transcription factor, its function is highly dependent on acetylation and phosphorylation levels [16; 17]. Rapid apoptosis of cell can be induced by phosphorylation of p53 [21]. Accordingly, oral cancer having aberrant p53 usually shows low levels of p53 acetylation and phosphorylation [3; 4]. In this study, 4 HR treatment increased the acetylation and phosphorylation of p53 in YD-15 cells (Figures 1 and 2). The expression levels of HDAC4 and HDAC activity were decreased upon 4 HR treatment (Figure 3). YD-15 cells have a p53 mutation in their DNA-binding domain [18]. The expression of Bax was increased upon 4 HR treatment in YD-15 cells (Figure 5). Apoptosis induced by 4 HR was confirmed by cytochrome c leakage in YD-15 cells after 4 HR treatment (Figure 4). In the xenograft model, 4 HR administration increased the survival rate, but the difference was not significant (Figure 6). A protein array was performed using the sera from xenografted animals. The phosphorylation of p53, as well as the expressions of MDM2 and MAPKAPK2 (MK2) were increased, but that of BRCA1, HDAC4, and HDAC5 were decreased (Table 1). Collectively, 4 HR treatment in oral cancer cells with a _TP53_ mutation reactivated p53 by acetylation and phosphorylation. Transcription factors are activated by acetylation and phosphorylation [22]. Accordingly, p53 activation and its translocation into the nucleus are achieved through p53 acetylation and phosphorylation [23]. Since histone deacetylase (HDAC) removes the acetyl group from the target protein, its inhibitor increases the acetylation of target proteins [9], and 4 HR is a class I and II HDAC inhibitor [13]. In this study, treatment with 4 HR increased the acetylation of p53 in YD-15 cells (Figure 2). Phosphorylation is also important for signal transduction [24]. Treatment with 4 HR also increased the phosphorylation of p53 in both YD-9 and YD-15 cells (Figures 1 and Supplementary S1). When _TP53_ expression was silenced by siRNA, the expression levels of Bax were still elevated compared to that in the siRNA control group (Figure 5). Some cancers show gain-of-function mutation in _TP53_[25]. The _TP53_ mutation in YD-15 cells might be gain-of-function mutation which might contribute to tumour survival. Accordingly, inhibiting _TP53_ expression by siRNA increased the expression levels of Bax, which is important for cellular apoptosis (Figure 5). Treatment with 4 HR increased the expression levels of Bad in the siRNA-_TP53_ group (Figure 5). Thus, 4 HR could increase the expression levels of Bad in both p53-dependent and -independent manners. Since 4 HR treatment increased ER and mitochondrial stress [14], the increased expression of Bad and Bax might be mediated by ER and mitochondrial stress. Administration of 4 HR in the xenograft model resulted in an improved survival rate (Figure 6B). However, the difference between the groups was not statistically significant \begin{table}
\begin{tabular}{c c c c} \hline
**Cancer Signal** & **Ratio*** & **p53 Signal** & **Ratio**\({}^{\ddagger}\) \\ \hline Caspase-9 & 1.539 & p-MK2 (Thr334) & 1.440 \\ Histone H2A & 1.286 & p-MDM2 (Ser166) & 1.218 \\ STAT4 & 1.434 & p-BRCA1 (Ser1524) & 0.777 \\ ERK8 & 2.836 & p-HDAC4 (Ser632) & 0.756 \\ HDAC4 & 0.766 & p-HDAC5 (Ser259) & 0.799 \\ MDM2 & 1.890 & p-p53 (Ser315) & 1.203 \\ \hline \end{tabular} \({}^{\star}\)4 HR/Control. \({}^{\dagger}\) phosphorylated (4 HR)/unphosphorylated (4 HR) \(\div\) phosphorylated (control)/unphosphorylated (control).  
\end{table} 
Table 1: The results of protein array performed using serum samples. (\(p>0.05\)). The main reason for the insignificant results might be due to small sample size. Although YD-15 cells have the mutant p53 protein, their ectopic tumour formation rate is notoriously low [18]. Despite 10 animals being included in each group, the success rate of tumour growth was very low (approximately 50%).