3.1. R248Q mutation of p53 amplified p-AKT signaling in ovarian cancer cells
We first investigated whether p53R248Q affected EGFR (a member of the RTK family) and its downstream signaling in OVCAR3 cells (HGSOC cell line with p53R248Q). Since EGFR is aberrantly overexpressed and activated in ovarian cancer [32, 33], we stimulated pre-starved cancer cells with epidermal growth factor (EGF) to activate the EGFR signaling. Both activated PI3K/AKT and MAPK/ERK pathways under EGF stimulation have been implicated in various cancers. Therefore, we examined these EGFR downstream markers under the condition of p53 siRNA knockdown. We found that p-AKT and p-MAPK decreased in OVCAR3 cells after p53R248Q knockdown (Supplementary Fig. S1A). Using an overexpression approach, we found that p-AKT was significantly upregulated after augmented expression of p53R248Q in OVCAR3 cells (Supplementary Fig. S1B). Taken together, these data indicated that the R248Q mutation of p53 amplified p-AKT signaling in OVCAR3 cells.
3.2. p53R248Q overexpression and EGF stimulation resulted in similar cytonuclear trafficking of AKT, EGFR, MDM2, and FOXO3a
AKT signaling is known to mediate intracellular trafficking of various receptors and regulatory proteins. We thus investigated how the R248Q mutation of p53 alters the intracellular molecular trafficking of EGFR, MDM2, and FOXO3a in association with AKT (Fig. 1).
We found that, after either EGF stimulation or p53R248Q overexpression, most of the AKT translocated into the nucleus completely. However, when p53R248Q overexpression was combined with EGF stimulation, some AKT remained in the cytoplasm (Fig. 1A). Consistent with past studies, our data implied that AKT enters the nucleus in response to growth factors to exert regulatory activities [34-36]; thus, we continued to investigate how AKT translocation correlates with the intracellular trafficking of EGFR and MDM2.
Similar but different from the aforementioned findings, after EGF stimulation or p53R248Q overexpression, both EGFR and MDM2 appeared to converge and translocate to the periphery of the cell nucleus (Fig. 1B and 1C). In comparison to the control group, we also observed some EGFR still localized on the cell membrane, while most of the MDM2 translocated to the periphery of the cell nucleus. Interestingly, when p53R248Q overexpression was combined with EGF stimulation, the EGFR located on the cell membrane and the MDM2 clustering around the nucleus were not detected, and most of the MDM2 translocated into the cell nucleus.
On the other hand, it has been reported that FOXO3a, a member of the forkhead box O (FoxO) transcription factor families and a tumor suppressor, accelerates its nuclear export when it is phosphorylated by nuclear AKT [37]. Therefore, we speculated that this tumorigenic marker is affected by p53R248Q. Based on our data, we found that FOXO3a, which mostly localized in the nucleus in the control group, distributed uniformly in the cytoplasm under the following conditions: after EGF stimulation, with p53R248Q overexpression, and p53R248Q overexpression combined with EGF stimulation (Fig. 1D).
These findings suggest that the R248Q mutation of p53 promotes tumorigenesis-related AKT signaling by affecting molecular intracellular trafficking.
3.3. Combined blockade by gefitinib and JNJ attenuated EGFR and MDM2 cytonuclear trafficking
The comparable translocation patterns of EGFR and MDM2 implied that the molecular cytonuclear trafficking may relate to the synergistic effect of combined EGFR and MDM2 inhibition that has been reported in our previous study [30]. In this present study, we again used gefitinib (EGFR tyrosine kinase inhibitor) and JNJ-26854165 (referred to as JNJ; MDM2 E3 ubiquitin ligase domain inhibitor) to determine whether such combined inhibition would alter the intracellular localization patterns of EGFR and MDM2.
By immunocytochemistry staining analysis, we first reconfirmed that both EGFR and MDM2 would translocate from the cytoplasm to the nucleus in accordance with each other over time after EGF stimulation (Supplementary Fig. S2). Next, we found that such nuclear convergence of EGFR was attenuated under treatment with gefitinib, JNJ, or both. Interestingly, the nuclear convergence of MDM2 was only disrupted under the combination treatment of JNJ and gefitinib (Fig. 2A and 2B). These results indicate that the combined inhibition of gefitinib and JNJ could prevent the converging nuclear translocation of EGFR and MDM2, which might regulate further signaling and oncogenic activity.
3.4. R248Q mutation of p53 increased the sensitivity of EGFR and MDM2 inhibitors
To further explore the impact of p53R248Q on the cellular response to combination therapy for HGSOC, we evaluated the anti-cancer efficacy of gefitinib and JNJ in OVCAR3 cells with different p53 statuses.
As shown in Supplementary Table 1, the combined inhibition of EGFR and MDM2 significantly reduced the IC50 values compared to the single agent treatment. Interestingly, the IC50 values of JNJ and gefitinib alone reduced to 66% (from 21.83 to 14.35 μM) and 78% (from 33.74 to 26.47 μM) when the cells were overexpressing p53R248Q. On the other hand, the combined inhibition resulted in a slight reduction (from 8.57 to 8.18 μM) of IC50.
For a more intuitive comparison, we summarized the data as Fig. 3 to highlight the significant reduction in effective drug concentration under combined inhibition. A synergistic effect was observed whether cells were pre-transfected with the p53R248Q plasmid or not (Fig. 3A, 3B). Interestingly, gefitinib and JNJ alone were more effective when OVCAR3 cells were pre-transfected with the p53R248Q plasmid (Fig. 3C, 3D). However, the combined inhibition effect remained similar whether OVCAR3 cells were pre-transfected with the p53R248Q plasmid or not (Fig. 3E). Taken together, these results suggest that the R248Q mutation of p53 increases the sensitivity of OVCAR3 cells to EGFR and MDM2 inhibition to various degrees.
3.5. R248Q mutation of p53 decreased the synergistic lethal effect of gefitinib and JNJ
To better evaluate the synergistic effects exerted by combined inhibition in different p53 statuses, we adapted the well-known Chou and Talalay’s combination index (CI) method to perform in-depth analysis. It should be noted that a CI value less than 1 is defined as drug synergism [31, 38].
The CI analysis revealed that in the absence of overexpression of p53R248Q, the area below the CI=1 line was larger than that of the p53R248Q overexpression group (Fig. 4A and 4B). We also generated isobolograms to quantify the drug synergism at different effective doses (EDs). In comparison to the p53R248Q overexpression group, the symbols (colored as specified) were farther from the corresponding colored lines, and the CI values were also lower in the absence of overexpression of p53R248Q (Supplementary Table 2, Fig. 4C, 4D). The CI values and the synergism grading are summarized in Supplementary Table 2.
In OVCAR3 cells overexpressing p53R248Q, the drug combination displayed slight synergism (CI=0.87) at ED50, but changed to moderate (CI=0.72) and enhanced synergism (CI=0.67) at ED75 and ED90. Interestingly, OVCAR3 cells without p53R248Q overexpression showed enhanced synergism (CI=0.65) at lower doses (ED50). This finding implied that a higher dose of JNJ and gefitinib (ED90) was required to reach synergism in OVCAR3 cells overexpressing p53R248Q. In contrast, lower dose of drug combination (i.e. ED50) exerted synergism in OVCAR3 cells without p53R248Q overexpression. Such data supports that overexpression of p53R248Q may attenuate the synergistic lethal effect of JNJ and gefitinib.
3.6. MAPK and p21 regulated the effects of single and combined treatment of gefitinib and JNJ
To provide more clues about the regulatory effect of p53R248Q on the observed differential sensitivity to JNJ and gefitinib, we analyzed the expression profiles of MAPK and p21 (a p53 target gene associated with cell cycle and apoptosis pathways) under different conditions of JNJ and gefitinib concentration and combination.
As expected, the enhanced expression of p53 was detected, which served to validate the overexpression of p53R248Q after transfection. Next, the results revealed that p-MAPK was significantly reduced by gefitinib in a dose-dependent manner (Fig. 5A). Moreover, at a dose of 10 μM, gefitinib exerted a stronger MAPK-reduction effect in p53R248Q-overexpressing cells compared to the cells without overexpression (Supplementary Fig. S3A). We also found that the expression level of p21 was significantly increased by JNJ in a dosage-dependent manner. Upon examination and comparison of the two DMSO control groups, we found that the expression level of p21 was lower in p53R248Q-overexpressing cells (Fig. 5B). However, with increasing JNJ dose, p21 increased significantly (approximately 17-fold) in p53R248Q-overexpressing cells compared to the cells without overexpression (approximately 4-fold) (Supplementary Fig. S3B). Strikingly, the effects were reversed when JNJ was combined with gefitinib (Fig. 5C, Supplementary Fig. S3C, S3D).
These results imply that in the presence of p53R248Q overexpression, the augmented sensitivity to JNJ or gefitinib and the weakened synergistic effect of combined treatment might be associated with the alternating expression of MAPK and p21.