In this study, we treated the HPV-positive OPSCC cell line HN26 with three proteasome inhibitors and one MDM2-inhibitor in combination with cisplatin. The main objective was to determine if there could be synergistic effects when combining cisplatin with the potentially p53 enhancing substances when treating HPV-positive tonsil cancer. Such synergistic effects have previously been documented in both uterine cervical cancer and HPV-positive head and neck squamous cell carcinoma cell lines [13, 23] and have been proposed based on the high proportion of wild-type TP53 in HPV-positive cancers and the importance of p53 removal by the HPV E6 protein for carcinogenesis.
In our experiments, all three proteasome inhibitors displayed antagonistic effects in combination with cisplatin while additive effects were seen with the MDM2 inhibitor. Thus, no synergy could be detected with any of the combinations. This implies that the combination of proteasome inhibition with cisplatin might result in negative effects for HPV positive OPSCC patients rather than improved treatment outcomes. Possibly this could be different for MDM2 inhibition, though only additive effects were detected.
To us, these findings were unexpected. Our original hypothesis was that the proteasome and MDM2 inhibitors would increase the expression of p53 leading to increased apoptosis and a synergistic effect on cell killing in combination with cisplatin. Instead, the effects on p53 protein expression were barely above the detection level of the Western blot for bortezomib, ixazomib and RG7112 and below it for carfilzomib (Figs. 3 and 4). This contrasted with an earlier study in which bortezomib induced a substantial increase in p53 expression in three HPV positive HNSCC cell lines . We also investigated if there was increased expression at other time points after treatment start for bortezomib, but the peak of expression was at 24 h also in combination with cisplatin (Fig. 3).
In line with the marginal effects on p53 expression, we could not detect any apoptosis as measured by PARP1 or caspase-3 cleavage. This was true for all substances and combinations with cisplatin (Fig. 4) – also tested at different time points for bortezomib (Fig. 3). Obviously, the expected increase in p53 mediated apoptosis leading to an enhanced effect of cisplatin did not occur in the investigated cell line. The reason for this cannot be deduced from our results, but the fact that the MDM2 inhibitor did not increase p53 expression and that p21 expression was increased by the proteasome inhibitors indicated that it was not failure to inhibit the proteasome activity that caused the lack of effect. An ubiquitin-independent E6 promoted degradation of p53 has been described and could possibly explain our results .
The TP53 gene in HN26 is homozygous for arginine at codon 72 which has been shown to make p53 more susceptible for E6 mediated degradation . Potentially, this might affect the ability of proteasome inhibition to substantially increase the levels of p53 protein. But on the other hand, at least one of the cell lines (UD-SCC-2) used by Li and Johnson  was shown to be homozygous for arginine at codon 72  while one of the other cell lines (UPCI:SCC090) was heterozygous for proline and arginine at this site , while still displaying bortezomib induced up-regulation of p53. Thus, rendering it unlikely that the effect on HN26 cells was due to arginine at position 72.
A large number of different proteins are regulated by proteasome degradation, for example, in apoptosis and cell cycle control. In the absence of p53 up-regulation with resulting increase in apoptosis as a response to proteasome inhibition, a multitude of different outcomes are therefore possible. We investigated if cell cycle progression was affected by proteasome inhibition in the HPV-positive cell line and found accumulation of cells in G1/G0 and fewer cells in S phase after incubation with all proteasome inhibitors, while incubation with cisplatin resulted in an increase of cells in S-phase. When combining bortezomib with cisplatin, the cisplatin induced S-phase increase was reversed (Fig. 5). Thus, a possible explanation for the reduced efficacy of cisplatin when combined with inhibitors could be the G1-arrest caused by the inhibitors, giving the cells time to repair cisplatin-induced DNA-damages before migration in the cell cycle, and hence becoming less prone to succumb from mitotic catastrophe.
Bortezomib and carfilzomib both induced a high increase in p21 expression, whereas ixazomib increased p21 in combination with cisplatin as compared with cells treated with cisplatin o alone. As p21 is regulated by proteasome degradation, this could be one mechanism responsible for the observed cell cycle alterations.
However, also the MDM2 inhibitor RG7112 affected cell cycle progression to an extent comparable with the proteasome inhibitors, although not antagonizing the effects of cisplatin (Fig. 5). Thus, cell cycle regulation per se does not seem to suffice in explaining the antagonistic effect of the proteasome inhibitors. As RG7112 did not affect p21 expression, neither with nor without cisplatin, one possibility is that other p21-regulated mechanisms are involved, or that other proteins that are affected by proteasome inhibition play an important role in HN26 cells.