Certain combination treatment regimens produce better response than the individual drugs alone [32]. Herein, we examined the combined effect of BRAFi with cisplatin in colorectal cancer cell lines and a mouse xenograft model. Firstly, the effect of cisplatin on CRC cell lines was analyzed. We found that following treatment of BRAF mutant cells with cisplatin, reduction of cell viability, G2/M phase arrest and phosphorylation of H2AX were observed. Cisplatin is a genotoxic drug inducing the formation of single-nucleotide damage of guanine (monoadducts), intrastrand and interstrand cross-links [33]. Monoadducts and intrastrand cross-links are repaired by the nucleotide excision repair (NER) mechanism [34], while the removal of interstrand cross-links requires the activation of several DNA repair pathways, such as homologous recombination, NER and translesion synthesis [35–37]. Of note, DNA double-strand breaks (DSBs) are produced as intermediates in the interstrand cross-links repair process [38]. Following the detection of DSBs, cells induce the phosphorylation of histone H2AX on serine 139 (γH2AX), by the apical signaling kinases ataxia telangiectasia mutated (ATM) and ATM-Rad3-related (ATR) [39]. Since the induction of γH2AX is an early event in the activation of the DNA damage response network, it is generally utilized as a marker of DSBs. Previous studies have also demonstrated that cisplatin impairs the synthesis of the DNA [40, 41], induces S-phase slowdown [42] and triggers a G2/M arrest through the inhibition of the CDK1/cyclin B activity [42, 43].
In addition, we found that following treatment of BRAF mutant cells with cisplatin, phosphorylation of the ERK1/2 kinase was observed. In accordance with our results, previous studies have shown that cisplatin treatment of HeLa cells caused a dose- and time-dependent phosphorylation, and therefore activation, of ERK, and that activation of this kinase is important for the induction of the apoptosis pathway via the cytochrome c release from mitochondria [44]. In contrast, Wei et al. [45] have shown that ERK signaling inhibition enhanced the susceptibility of ovarian cancer cells to cisplatin. Another report has shown that various genotoxic insults, such as adriamycin, ultraviolet irradiation, etoposide and ionizing radiation induced activation of ERK1/2 in the MEF and IMR90 primary cells, the NIH3T3 immortalized cells and the transformed MCF-7 cells, thus leading to cell cycle arrest or induction of apoptosis, depending on the drug concentration used [46].
Next, the effect of BRAF inhibition on CRC cells was analyzed. We found that treatment of BRAF mutant cells with a BRAFi resulted in decreased cell viability and reduction of ERK activation/phosphorylation. As expected, the inhibition of BRAF had no effect on cell viability of BRAFwt cells. It is known that the pathway that leads to the activation of the two isoforms of ERK (ERK1 and ERK2) is initiated after ligand binding to a plasma membrane receptor tyrosine kinase and the activation of the GTP-binding protein Ras. Then, Ras activates the MAP3K kinase Raf, followed by the activation of the mitogen-activated protein kinase kinase (MEK or MAP2K), which in turn phosphorylates threonine and tyrosine residues in the Thr-Glu-Tyr (TEY) sequence of ERK1/2 [47]. In several cancer types, such as melanoma, hairy cell leukemia, colon carcer and papillary thyroid carcinoma, a mutation in codon 600 of exon 15 (V600E) has been reported. This mutation has been implicated in various mechanisms of cancer progression, such as stimulation of the MEK/ERK pathway, prevention of immune response, avoidance of apoptosis and senescence, angiogenesis, tissue invasion and metastasis [48]. Interestingly, the inhibition of the BRAF kinase results in decreased pERK activity and the reduction of cell proliferation, indicating that the decreased pERK activity can be used as a pharmacodynamic biomarker of the BRAF inhibition.
Moreover, we found that treatment of BRAF mutant cells with a BRAFi resulted in perturbation of cell cycle progression, inducing a G0/G1 arrest. An accumulating body of evidence suggests that the ERK kinases are involved in the cell cycle progression from G1 to S phase, which occurs immediately after growth factor stimulation. Indeed, previous studies have shown that ERK inhibition by a MEK inhibitor that was given even immediately prior to the beginning of the S phase, blocked cell cycle entry into the S phase [49]. That is, after growth factor stimulation, the activation of ERK causes the phosphorylation, and thus activation of the ETS transcription factor Elk-1 and results in the upregulation of immediate-early genes, such as the proto-oncogene c-fos, the expression of which is involved in the induction of delayed-early genes, such as cyclin D [50–52]. Then, the Cyclin D/Cdk4 complex initiates the phosphorylation of retinoblastoma, a protein known to activate the E2F family of transcription factors, and regulates the expression of several target genes, such as cyclin E. Next, the complex of cyclin E with Cdk2 further phosphorylates the retinoblastoma protein, and activates the E2F transcription factors. These sequential events regulate the synthesis of various proteins that are involved in the entry of the cells in the S phase [53]. Previous studies have revealed a set of genes whose expression levels were rapidly reduced after ERK inactivation. Interestingly, several of these genes were found to possess antiproliferative properties, i.e. they have the ability to suppress the entry of the cells into the S phase. Together, these data suggest that the BRAF inhibitor-induced ERK inactivation blocks S phase entry [49, 54].
Also, in accordance with our previous study [29], herein we found that following treatment of BRAF mutant cells with a BRAFi, phosphorylation of H2AX was observed. In fact, evidence has accumulated that histone H2AX phosphorylated on Ser-139 (γH2AX), in addition to be a critical component of the DSB repair mechanism, is also implicated in many other biological processes. For example, Fragkos et al. [55] have shown that, in the absence of DNA damage, phosphorylation of H2AX is an early sign of replication stalling, inhibiting cell cycle progression from the G1 to the S phase. Interestingly, they found that after inhibition of the replication machinery, γH2AX is needed for increasing the levels of p21, thus resulting in checkpoint activation and cell cycle arrest. These results suggest that the phosphorylation of H2AX on Ser-139 that was observed in the present study correlated with the G0/G1 arrest after treatment of BRAF mutant cells with a BRAFi. Although the exact mechanism of the interaction between γH2AX and the p53/p21 pathway are still unknown, it is possible that H2AX phosphorylation affects the interaction of PCNA with chromatin, allowing the stable binding of PCNA to p21 and blocking its ubiquitination [56].
Importantly, the antitumor activity of the combination treatment with cisplatin and PLX4720 was further validated in vivo in mouse xenografts of RKO cells, with the combinatorial treatment showing superior therapeutic potential than each drug alone. Combinatorial treatments of PLX4720 and other therapeutic compounds have proven efficient in growth suppression of distinct tumor types [57, 58]. Especially in the case of colorectal cancer, in which Vemurafenib monotherapy is of no appreciable value for patients [59], combination of the RAS/MAPK pathway inhibition and chemotherapy could potentially ameliorate the observed resistance mechanisms that contribute to reactivation of cancer cell proliferation. Finally, drug combinations can achieve higher therapeutic responses in lower individual doses, thus avoiding the complications of off-target toxicities induced by high drug concentrations. Herein, the in vivo confirmation of the remarkable in vitro results paves the way for the exploitation of similar therapeutic combinations in multiple preclinical and potentially, clinical settings. However, more in vivo studies regarding pharmacokinetics and other pharmacological parameters are required in order to accurately characterize the therapeutic value of the proposed regime.
Taken together, our study demonstrates that the combined treatment with BRAFi and cisplatin is more effective than single-drug treatment in preclinical models in vitro and in vivo (Fig. 6), suggesting that these data, once further validated at the preclinical level, can be exploited for the design of new therapies for the treatment of CRC.