Establishment and phenotype of the drug resistance model.
To investigate the molecular mechanisms underlying BRAFi resistance, we exposed the melanoma cell line A2058 harboring the BRAF V600E mutation to high concentrations of vemurafenib. After 6 months, we obtained the resistant cell line A2058R. We found that the cells became slender under the action of vemurafenib at a high concentration (Figure 1A, 1B). Results of the CCK8 experiment confirmed that the IC50 of drug-resistant A2058R cells was higher than that of primary A2058 cells, (A2058 IC50 = 0.71, A2058R IC50 = 21.95, RI = 30.9, P < 0.005) (Figure 1C, 1D). Scratch and Transwell assays confirmed that A2058R cells have greater invasion and migration abilities than A2058 cells (Figure 1E – 1I). Meanwhile, WB assays showed that the levels of CD271, SOX10, TWIST, and SLUG were elevated in A2058R cells, indicating that they possess higher stemness and EMT abilities.
PCK1 protein and AKT/PI3K signaling were key factors in the transcriptome sequencing results.
After normalizing the transcriptome sequencing results (Figure 2A), we found that there were differences in the gene expression profiles among the four samples (Figure 2B), which was further corroborated by PCA results (Figure 2C). Differential expression analysis was performed, and we focused on PCK1, a gene whose expression level is high and constant in R1, R2, and R3, and we considered it as the key target gene for conferring of drug resistance (Figure 2D). In KEGG enrichment analysis (Figure 2E), we observed that the PI3K/AKT signaling pathway molecules were continuously highly expressed in the three drug-resistant samples. Our results in combination with the literature on this topic indicate that the PCK1 and PI3K/Akt signaling pathways are involved in the process of acquired drug resistance induced by BRAFi in melanoma cells. Furthermore, western blot analysis showed that the expression of the PCK1 and PI3K/Akt signaling pathways was up-regulated and activated in drug-resistant cells. In addition, when we inhibited the PI3K/Akt pathway, the expression of PCK1 decreased, suggesting that PCK1 is a crucial downstream molecule for the PI3K/Akt signaling pathway for achievement of drug resistance (Figure 2F).
PCK1 activity determined melanoma cell resistance to vemurafenib.
Next, we constructed low expression and overexpression PCK1 cell lines using siRNA and overexpression viruses, respectively. Western blot results indicated that the transfection was successful (Figure 3A). After PCK1 knockdown, cell viability significantly reduced; thus, the IC50 of siPCK1 and the resistance index (RI) both decreased (RI = 4.46), P < 0.005 (Figure 3B, 3C).
However, the cell viability and resistance index (RI) were higher (RI = 9.27), P < 0.005, in the cell line overexpressing PCK1 (Figure 3B, 3D). Therefore, the results indicated that PCK1 is a crucial molecule that can enhance the tolerance of melanoma cells to vemurafenib.
PCK1 promoted the proliferation, migration, and stemness of melanoma cells with V600E mutation.
Based on the hypothesis that the development of drug resistance may be caused by related genes that induce cancer stemness, EMT, and apoptosis inhibition, we first designed scratch and invasion experiments and found that the PCK1 gene status was related to the proliferation and invasion of melanoma cells, while the activation of PCK1 could promote drug resistance in melanoma and increase the severity of the disease (Figure 4A, 4B). Next, flow cytometry was conducted, and the results showed that the knockdown and overexpression of PCK1 did not have a direct effect on apoptosis, although statistical differences were noted. This suggested that PCK1 might indirectly induce BRAFi resistance through PPP (Figure 4C–4G). In addition, the results of the tumor spheroidization test on a low adhesion plate showed that the spheroidization ability of A2058R cells was significantly higher than that of A2058 cells (Figure 4H), that of the SiPCK1 group was decreased, and that of the OE group was restored after the overexpression of PCK1. Combined with the western blot results, these results suggested that CD271 and SOX10, which have been recognized as indicators of melanoma stemness, were highly expressed in A2058R and OE cells (Figure 4I), indicating that PCK1 could modify cancer stemness in the development of drug resistance. Meanwhile, in this experiment, PCK1 could not alter the EMT of melanoma for achievement of drug resistance (Figure 4J). These lines of evidence illustrated that the activation of PCK1 potentiated melanoma proliferation, migration, and tumor stemness, all the phenotypes associated with drug resistance. The results revealed that PCK1 did not contribute to drug resistance directly by inhibiting apoptosis but, most likely, contributed indirectly to acquired drug resistance by triggering some of the cascades of the biochemical metabolic chain.
Synergistic effect of PCK1 inhibitor 3-MPA combined with vemurafenib on drug-resistant cells.
A CCK8 assay was used to detect the activity of A2058R cells under different drug treatment regimens to evaluate the advantages of a combined treatment strategy. The IC50 of A2058R cells treated with 3-MPA or vemurafenib alone did not change significantly (Figure 5A, 5C). However, the IC50 of 3-MPA combined with vemurafenib was significantly lower than that of vemurafenib alone, which indicated that 3-MPA increased the resistance inhibition of vemurafenib (Figure 5B, 5C). In order to evaluate the efficacy of the combination therapy, we used the Chou-Talalay method to compare the best fitting IC50 of each treatment group again. In the experiments in which drug-resistant A2058R cells were treated, the IC50 of the 3-MPA + vemurafenib group was 6.37, that of vemurafenib monotherapy was 14.47, that of 3-MPA monotherapy was 27.47, and the combination index (CI) = 0.3362 < 1, which showed that the combination of 3-MPA and vemurafenib had a synergistic effect on pharmacology (Figure 5D, 5E). Moreover, only when the reaction part of the system reaches an 0.8 < FA < 0.9, the synergistic effect will be reversed to the antagonistic effect, which indicates that change in the doses of the two drugs has little effect on the synergistic effect and that this effect is stable (Figure 5F).
Inhibition of PCK1 by 3-MPA led to ROS accumulation and oxidative damage in drug-resistant cells.
Since PCK1 is a key enzyme in the PPP process and PPP controls the intracellular ROS levels, the supplement of reduction equivalent is undoubtedly of great value to melanoma cells. Therefore, we speculated that the downstream mechanism of PCK1 might be related to the regulation of ROS levels. We used the High Content Live Cell Imaging System to observe the ROS levels. As shown in Figure 6A, we found that the ROS level in primary A2058 cells was significantly higher than that in A2058R cells (Figure 6B), which demonstrated that drug-resistant cells have a better ROS reduction ability. When A2058R cells were exposed to vemurafenib, the ROS level was still lower than that in A2058 cells. In addition, the ROS levels of primary A2058 cells and drug-resistant A2058R cells were higher than those in A2058R cells after adding 3-MPA. When A2058 and A2058R cells were treated with a combination of 3-MPA and vemurafenib, the ROS level was increased significantly, which suggested that the ROS level of drug-resistant cells was determined by the activity of PCK1.
We generated a subcutaneous xenograft model from nude mice, forming cryosections that were analyzed using a ROS probe (Figure 6C). Combined with the tumor volume/wt (Figure 7D, 7E), we found that the ROS levels were higher in the A2058 + vemurafenib group than in the A2058 + DMSO group, whereas the number of tumor cells was reduced relative to the A2058 + DMSO group, indicating that vemurafenib inhibited V600E mutated melanoma progression by generating strong oxidative damage. Compared with the A2058R + vemurafenib group, the A2058 + vemurafenib group exhibited higher intracellular ROS levels and decreased tumor cell numbers, indicating that the drug-resistant cells hedge the killing effect of vemurafenib via metabolic reprogramming. Compared with the A2058R + vemurafenib group, the A2058R + 3-MPA + vemurafenib group showed a significant increase in ROS levels and a decrease in the number of tumor cells, demonstrating that, in drug-resistant cells, the inhibition of PCK1 sensitizes melanoma to BRAFi inhibition. The increase in ROS levels was mainly caused by the obstruction of the PPP pathway. However, when we added 3-MPA (20 μmol/l) to A2058 and A2058R cells, the WB results showed that the expression of KEAP1 was increased after PCK1 silencing, which indicated that PCK1 inhibited the expression of KEAP1 (Figure 6D). These pieces of evidence all pointed the spearhead of resistance toward ROS reduction, which was the result of PCK1 activity. 3-MPA blocked PCK1, thereby enabling ROS accumulation and suppressing chemoresistance in melanoma cells.
3-MPA improved the inhibitory effect of BRAFi in vivo
Next, we evaluated the effect of combination therapy in melanoma using a subcutaneous xenograft mouse model (Figure 7A, 7B). According to the wt and volume data (Figure 7C, 7D), we found that the combination of vemurafenib and 3-MPA in the drug-resistant group could reverse the drug-resistant situation and significantly reduce the wet and volume of the tumor tissue. Compared with A2058 primary melanoma cells, the inhibition ability of the combination group was even more prominent, which suggested that the combination of BRAFi and PCK1 could achieve an excellent inhibition effect in BRAFi-resistant cells with PCK1 mutation. Combined with the immunohistochemical results, as mentioned above, we further confirmed that the main effect of 3-MPA was to increase the ROS levels of melanoma cells, disrupt the oxidative balance in drug-resistant melanoma cells, and expose them to an oxidative attack again.
The immunohistochemistry results showed that in the subcutaneous tumor tissues of nude mice inoculated with primary A2058 cells, the expression of PCK1 in the vemurafenib group was up-regulated compared with the DMSO group, while KEAP1 was inhibited (Figure 6D). In the tissues of A2058R cells, the up-regulated expression of PCK1 in the group treated with vemurafenib in combination with 3-MPA was limited compared with that in the vemurafenib group, but the expression of KEAP1 was up-regulated. Therefore, both the in vitro and in vivo experiments confirmed that vemurafenib could induce high expression of PCK1, and then inhibit the synthesis of KEAP1, thus inducing drug resistance. However, when 3-MPA was used to inhibit PCK1, KEAP1 was also activated and ROS accumulated in the cells, which made the drug-resistant melanoma cells suffer from oxidative attack and inhibited tumor progression.