MEK and PI3K pathways are the two essential kinase cascades frequently dysregulated in various cancers, including human lung cancer [17, 19, 30, 31]. Both the pathways represent important signal transduction mechanisms that facilitate the proliferation and survival of cancers driven by growth factor receptors, such as EGFR. In the present study, we confirmed that treatment with a MEK162/BKM120 combination effectively inhibited cell proliferation, induced apoptosis, and G0/G1 cell cycle arrest in EGFR-TKI resistant NSCLC cell lines: H1975, H460, and A549. However, the treatment of cells with MEK162 or BKM120 alone was ineffective. The cytotoxic effects of the combination treatment can be attributed to the inhibition of both the MEK and PI3K pathways. Increasing evidence demonstrates that the MEK and PI3K signaling pathways may interact to promote the growth and survival of tumor cells [16, 32]. Also, EGFR activates MET through MAPK [33]. Therefore, the inefficacy of treatment with MEK162 or BKM120 alone can be attributed to the compensating cross-talk between the pathways. We also detected that the treatment with MEK162 elevated the phosphorylation of AKT in all three tested cell lines, which can be abrogated by a MEK162/BKM120 combination. In addition, a MEK162/BKM120 combination caused a decrease in the levels of p-ERK1/2, p-AKT, p-S6, and p-4E-BP1 in all three cell lines. Collectively, our data suggested that the synergistic inhibition of MEK and PI3K pathways, rather than selective inhibition of each pathway, achieved sufficient cytotoxic effects in EGFR-TKI resistant NSCLC cell lines, H1975, H460, and A549. These results corroborate the observation by Yao et al. [27] and Qu et al. [28].
It has been reported that in the MAPK pathway, activated RAF phosphorylates MEK, a dual-specificity tyrosine, and serine/threonine kinase, which predominantly activates the serine/threonine kinases, ERK1 and ERK2. ERKs are known to have key roles in controlling cell cycle regulation, proliferation, and survival [34, 35]. In the PI3K pathway, activated PI3K ultimately leads to the stimulation of AKT, which promotes cell survival by phosphorylating MDM2 (a negative regulator of the p53 tumor suppressor) and negatively regulating the pro-apoptotic Bcl-2 family members, BAD and BAX, and forkhead transcription factors, such as forkhead box O (FOXO). Activated AKT also negatively regulates the tuberous sclerosis protein complex 1 (TSC1) and TSC2, which leads to the activation of mTOR complex 1 (mTORC1), a key regulator of cellular growth and protein synthesis. MAPK pathway regulates mTOR signaling through the inactivation of the TSC1/TSC2 heterodimeric complex by ERK [35, 36]. In addition, a recent study demonstrated that inhibition of mTORC1 with everolimus leads to the activation of the MAPK pathway [36]. This phenomenon may be attributed to the theory that mTORC1 inhibition releases the "brake" on PI3K triggered by S6K, which activates the MAPK signaling. Therefore, we firmly speculate that the interactions between the two signaling pathways underlie the need to block both these pathways to achieve a significant inhibition of tumor growth, especially in EGFR-TKI resistant NSCLC.
It was also observed that the synergistic effects of the dual inhibition of upstream signal EGFR/MET and downstream signal MEK/PI3K on the NSCLC were profound regarding growth inhibition, apoptosis, cell cycle arrest, and expression of phosphorylated pathways. However, the observed effects differed among the three NSCLC cell lines tested depending on their genetic alterations. The H1975 cells were mostly sensitive to the BIBW2992 and ARQ197 combination, and H460 cells to the MEK162/BKM120 combination. The rational explanation might be that the H1975 cells were addicted to EGFR (T790M) and PIK3CA mutation and MET amplification, and H460 cells were addicted to K-RAS and PIK3CA mutation. In a previous study, we demonstrated that the BIBW2992 and ARQ197 combination downregulated the expression of phosphorylated ERK and AKT in H1975 cells [22]. Moreover, as the BIBW2992/ARQ197 combination was cytotoxic, it was observed that the treatment of H1975 cells with this combination exhibited little effect in the decreased phosphorylation of ERK and AKT in comparison to the treatment with the MEK162/BKM120 combination. The study speculated that other signaling pathways that serve downstream of EGFR T790M mutation and MET amplification whose contribution to malignant proliferation are larger than MEK/PI3K. Indeed, the network of signal transduction pathways in NSCLC cells is highly sophisticated. This warrants transcriptome and proteomic profiling under varied physiological conditions to elucidate the exact mechanisms of the mode of action of the therapeutics employed. Therefore, the observed effects of the MEK/PI3K inhibition necessitate further evaluation. In this study, the cytotoxicity of MEK162 combined with BKM120 was confirmed by MTT assay, cell cycle detection and Annexin V-PI double staining detection. Annexin V-PI double staining is a recognized method for detecting apoptosis, especially early apoptosis, with high accuracy. After the cytotoxicity of MEK162 combined with BKM120 on a first-generation EGFR-TKI resistant NSCLC cell line was confirmed, our next study focused on analyzing the effect of the drug on the cell signal transduction pathway, rather than on the expression of apoptotic effector molecules. Therefore, the expression of apoptotic effector molecules such as caspase was not detected. Of course, if time, energy and funds allow, we can carry out related work in the follow-up study to enrich the research results.
The innovation points of this study are as follows: (1) NSCLC cell lines resistant to first-generation EGFR-TKI with different genetic mutation background were used as the research subjects to compare the differences in cytotoxicity, apoptosis and expression of important transduction signal factors between combined inhibition of upstream pathway (EGFR/MET) and combined inhibition of downstream pathway (MEK/PI3K). To increase the basis for preclinical studies on the problem of choosing the combined inhibition of upstream signals located in the cell membrane or the combined inhibition of downstream signals located in the cell after the acquired drug resistance of molecular targeted therapy of NSCLC. This is the most important innovation of this study. (2) At the time of this study (2019), there were more MEK/PI3K inhibitors, but few studies have revealed which agent is less clinically toxic, and the corresponding clinical studies are also few. On the basis of reading a lot of literature, we chose MEK162 and BKM120 as the drugs to be used in the study, mainly because of their good effects and low toxicity. In fact, now more and more evidence shows that our judgment at that time is correct, and clinical studies on MEK162 and BKM120 are also increasing gradually. It shows a good application prospect, especially for MEK162. Our results add to the evidence of efficacy in preclinical studies of these drugs.
Figures 4 and 6 highlight the role of combined inhibition of the upstream pathway EGFR/MET and the downstream pathway MEK/PI3K in each cell line. Both our preliminary experiments and a large number of published papers have confirmed that monotherapy is ineffective in these cell lines, and it has been shown in Figs. 1 and 2 that monotherapy of BKM120 or MEK162 is ineffective in all three cell lines. Therefore, both for theoretical deduction and for saving money and energy in practical application, Results and images of monotherapy of the three cell lines were not shown separately.
In conclusion, dual targeting of MEK and PI3K effectively inhibited EGFR-TKI resistant NSCLC proliferation, promoted cell apoptosis, and induced cell cycle arrest. Therefore, the data generated provide a scientific rationale for co-targeting MEK/PI3K signaling as a strategy for EGFR-TKI resistant NSCLC, especially in K-RAS and PIK3CA mutation in NSCLC.