3.1 PARP inhibitor olaparib upregulates HR and PD-L1 in OSCC cells
The IC50 values of olaparib in Cal27 and Scc25 cells were 36 µM and 98 µM, respectively, indicating the sensitivity of Cal27 cells to drug stronger than Scc25 cells. (Figure_2a)Then the CCK8 assays also verified that hypothesis. (Figure_1a)To examine the possible role of HR in reaction to olaparib, treating cells with olaparib at various concentrations and evaluated levels of RAD51, BRCA1, TOPBP1, H2AX and phosphorylated γ-H2AX. In Cal27 cells, 5 µM olaparib treatment resulted in increased RAD51, BRCA1, and TOPBP1 and decreased γ-H2AX phosphorylation, H2AX. Similar results were observed in response to 10 µM, although to a lesser extent compared with 5 µM olaparib. In Scc25 cells, olaparib at 10 µM or 20 µM upregulated the expression of RAD51, BRCA1, TOPBP1 and γ-H2AX. (Figure_1b)
To further explore the mechanism of olaparib resistance in OSCC, we examined the PD-L1 expression. PD-L1 was consistent with HR-related proteins (RAD51, BRCA1, TOPBP1, γ-H2AX). (Figure_1b) We also examined the mRNA and protein expressions of HR genes RAD51 and CD274. (Figure_1c) Moreover, BRD4 expression was also upregulated by olaparib, which help explain the relationality of using BET inhibitor to augment treatment effect. (Figure_1a)
Together these results indicated that the PARP inhibitor olaparib induced the HR-mediated DNA damage response as well as high PD-L1 expression.
3.2 BET inhibitor and PARP inhibitor synergistically suppress cell proliferation
We next examined the effects of treating cells with olaparib, the BET inhibitor JQ1, or combining both inhibitors. Both olaparib and JQ1 at low concentrations had moderate impact on decreasing cell proliferation and viability of Cal27 and Scc25 cells when they were used alone. However, olaparib together with JQ1 greatly triggered cell death. The IC50 values of olaparib were significantly reduced after combination treatment (**P < 0.01) (Figure_2a), indicating that OSCC cells were more sensitive to olaparib. CI calculations confirmed that the combination JQ1 and olaparib treatment exhibited a synergistic effect (Table 1).
The JQ1 and olaparib combination treatment also caused colony formation inhibition compared with other groups. (Figure_2b) The action of the combined treatment on proliferation was tested using CCK8 assays. The results showed that combination of two drugs cause a remarkable decline in cells’ proliferation, comparing with individual inhibitors (**P < 0.01, Figure_2c).
These results demonstrated a synergistic inhibitory effect of two drugs on tumor cell viability in OSCC.
3.3 BET inhibitor and PARP inhibitor synergistically induce apoptosis, which correlates with higher p53 levels
We next examined whether the combination treatment affected the apoptosis of OSCC cells. Western blot analysis uncovered that the cleaved caspase-3 which is an important apoptosis marker was activated at a dramatically higher level in Cal27 and Scc25 cells after treating with JQ1 and olaparib. (**P < 0.01, Figure_3a, 3b)Our research also evaluated cell apoptosis by Annexin V-PE staining followed by flow cytometry in OSCC cells treated with variable inhibitors for 2 days. Apoptotic cells accounted for higher proportions in response to the combination treatment than treatment by either inhibitor alone. (**P < 0.01, Figure_3c, 3d)
P53 signaling is a key pathway that induces apoptosis. We thus next examined whether apoptosis was mediated by the p53 pathway. The combination treatment led to prominently increased p53 and phosphorylated p53 expressions compared with single treatments. (*P < 0.05, **P < 0.01, Figure_3a, 3b)
Together our findings suggest that JQ1 and olaparib exhibit synergistic effects on suppressing viability of OSCC cells, which correlated with activation of the p53 pathway.
3.4 BET inhibitor and PARP inhibitor co-treatment results in cell cycle arrest
Then we focused on the effects of the combination treatment on cell cycle in OSCC cells. The results are displayed in Figure. 4a and quantitative analysis is shown in Figure. 4b. JQ1 and olaparib alone resulted in G1 or G2/M phase cell cycle arrest, respectively, while the combination treatment caused G1 and G2/M phase arrest as well as a significant decline in S phase cells.
3.5 BET inhibitor and PARP inhibitor synergistically suppress the growth of tumors in vivo
Cal27 cells were injected into Six-week-old female mice and after the tumor volume reached approximately 150–250 mm3, mice were randomized into groups and treated with different inhibitors. The combination of JQ1 and olaparib potently inhibited tumor growth including tumor volume and tumor weight, comparing with single inhibitors used alone (**P < 0.01, Figure_5a, 5b, 5c). The results demonstrated that combined treatment exhibits better antitumor effects in OSCC in vivo compared with JQ1 or olaparib treatment alone.
3.6 BET inhibitor and PARP inhibitor combination suppresses HR mediated by ATR/CHK1 pathway
To explore the potential mechanism of the enhanced efficacy with combined treatment in OSCC, we examined several key DNA damage response factors involved in HR. Western blot and Immunofluorescence assay showed that these HR-related molecules were highly downregulated by the combination treatment compared with olaparib alone, although the expressions were slightly higher than in response to JQ1 alone. (Figure_6a, 6c) We also observed decreased RAD51 mRNA level in Cal27 and Scc25 cells treated with the combination therapy compared with olaparib monotherapy (**P < 0.01, Figure_6b), which verified that JQ1 combined with olaparib can act by suppressing HR.
ATR/CHK1 plays a critical role in the regulation of replication and is essential in HR, and thus we subsequently examined the ATR/CHK1 pathway. Consistent with our above results, the ATR/CHK1 pathway was increased in response to olaparib compared with controls, while the combination treatment markedly blocked this pathway compared with olaparib alone (**P < 0.01, Figure_6a, 6b), further indicating that JQ1 maximized olaparib’s effect through impairing HR mediated by ATR/CHK1 pathway inhibition.
These results indicate that the BET inhibitor JQ1 disrupted HR via inducing ATR/CHK1 inhibition, thus sensitizing the OSCC cell response to the PARP inhibitor.
3.7 BET inhibitor and PARP inhibitor combination attenuated expression levels of BRD4 and PD-L1
We continued to explore the mechanism behind the combinatorial synergism of JQ1 and olaparib in OSCC cells. The combination of JQ1 and olaparib reduced BRD4 expression to a greater extent compared with olaparib or JQ1 monotherapy in Cal27 and Scc25 cells, which verified that the sensitivity of OSCC to the BET inhibitor was also enhanced by the combination treatment. (*P < 0.05, **P < 0.01) (Figure_7a, 7b, 7c)
We next determined whether BRD4 correlates with PD-L1 expression in OSCC by examining a panel of OSCC cell lines. We observed a trend towards a positive relationship between BRD4 and PD-L1 expression. (Figure_7a, 7b) We then examined whether BRD4 and CD274 expression was also positively correlated in OSCC cells. Indeed, there was a significant positive mRNA correlation between BRD4 and CD274 in Cal27 and Scc25 cells treated with JQ1, olaparib or the combination. (Figure_7c) Notably, decreasing PD-L1 expression may be a strategy to optimize the antitumor effects of PARP inhibitors by adding BET inhibition.
Our results showed that the PARP inhibitor upregulated PD-L1 and BRD4 in OSCC cells, while BET blockade attenuated PD-L1-mediated immunosuppression activity, resulting in better treatment effect in OSCC cells. These results verified that the sensitivity of OSCC to the BET inhibitor was enhanced by the combination treatment with the PARP inhibitor.