Cisplatin based therapy: the role of the mitogen activated protein kinase signaling pathway

Cisplatin

Cisplatin is the first platinum drug approved globally and has been used as a drug for cancer chemotherapy for more than 30 years [1]. Cytostatic property of cisplatin was discovered by Barnett Rosenberg in the late 1960s when he observed anti-proliferative effect of platinum electrodes on bacterial suspension. Cisplatin has gained clinical success and has been proven to target various kinds of cancers, namely, lung, head and neck, bladder, cervical, and ovarian cancers [2].

Upon cisplatin entry to the cell, it becomes activated by hydrolysis. Chloride ions are displaced by water molecules resulting in a strong positive electrophile that covalently binds to any nucleophile such as nitrogen in purine residues. The most reported reaction is the 1,2-intrastrand cross-linkage of the activated cisplatin with the purine residues at nitrogen (N7) which was demonstrated in vitro in DNA of cisplatin-treated salmon sperm cells. These results were further approved in another in vivo experiment that analyzed white blood cells of cancer patients [15, 16]. Cisplatin causes damage in the DNA leading to p53 (tumor suppressor gene) activation. Meanwhile, damaged DNA is subjected to repair via p21 mediated cell cycle arrest. When the damaged DNA is not repairable, p53 induces apoptosis by inhibition of Bcl2 and consequent caspase activation [17]. The main mechanism of cisplatin-induced cancer cell death is via apoptosis. Apoptosis is a procedure of programmed cell death, and is generally manifested by distinct morphological changes to the cell, e.g. cell shrinkage, chromatin condensation, plasma membrane “budding”, exposure of phosphatidyl serine at the cell surface, and caspase activation [18]. The stimulation of a family of cysteines, otherwise known as caspases, is a critical step during the beginning stages of apoptosis. Caspases are characterized as either initiators or executioners of this cell death mechanism. The activation of caspases is dependent upon several stimuli, e.g. caspase-8 (initiator), activated by plasma membrane death receptors (DR), and caspase-9 (initiator), associated with mitochondrial collapse, resulting in caspase-3 and -7 (executioners) activation [19]. Executioner caspases are responsible for many of the biochemical activities contributing towards the induction of apoptosis. This includes poly (ADP-ribose) polymerase (PARP) cleavage and activation, which leads to DNA fragmentation. Apoptosis is carried out by two major pathways known as the extrinsic and intrinsic pathways [20]. The extrinsic pathway begins when ligands bind to tumor necrosis factor-α (TNFα) receptor family leading to the enrollment of procaspase-8 by adaptor molecules, resulting in the development of death-inducing signaling complex (DISC) [21]. However, the intrinsic pathway commences when the cell undergo stress such as DNA damage, which subsequently leads to mitochondrial cytochrome c release and interaction with activating factor-1 (APAF-1), to form an active apoptosome structure, which thereby activates procaspase 9. In response, and in order to regulate DNA damage induced by apoptosis, Bcl-2 family proteins are regulated and undergo several modulations via controlling the release of cytochrome c. Therefore, genotoxic stress induced by cisplatin can result in the activation of several signal transduction pathways, contributing towards the induction of apoptosis.

Strategies to overcome cisplatin resistance and toxicity

Although cisplatin is widely used anticancer drug, significant challenges still remain in regards to cisplatin resistance and cisplatin induced toxicity. There are different mechanisms attributed towards tumor resistance to cisplatin.

Cisplatin is introduced into cells either through passive diffusion or via transporters (copper transporter, CTR1) [3]. Previous studies have reported that loss of CTR1 resulted in less platinum entering cells and, consequently, drug resistance [22, 23]. Another reason for cisplatin resistance is due to the inactivation of active cisplatin by glutathione or metallothionein present in the cytoplasm of cells. These species are rich in sulphur containing amino acids like cysteine and methionine and leads to inactivation of platinum by binding to sulphur. Active efflux of platinum from the cells through copper exporters (ATP7A and ATP7B) and excretion through glutathione S-conjugate export GS-X pump, also contribute towards cisplatin resistance. Increased DNA repair mechanisms and downregulation of apoptotic pathways are other factors mediating cisplatin resistance [3].

Treatment with cisplatin has been associated with several toxic side effects including nephrotoxicity, oxidative damage to liver, cardiomyopathy, allergic reactions, ototoxicity and gastrotoxicity [15, 24–26].

Cisplatin resistance to tumor cells can be overcome by adopting techniques (Fig. 1) to improve the delivery of cisplatin to the target DNA. This can be achieved by administering cisplatin in polymers or liposomes, and by inhibiting levels of species (glutathione and metallothionein) which inactivate cisplatin and thereby decrease its antitumor efficiency [3]. Recent in vitro and in vivo studies demonstrated that nanoscale micelles encapsulating ethacraplatin, a conjugate of cisplatin, and ethacrynic acid (inhibitor of glutathione S-transferase) resulted in enhanced accumulation of active cisplatin in cancer cells by inhibiting glutathion S transferase and circumventing deactivation of cisplatin [27]. Cisplatin resistance can also be controlled by using antisense oligodeoxynucleotides (ODN) targeting responsible oncogenes for cisplatin resistance, where the ODN hybridize with the target mRNA and prevents its translation; or by inactivation of oncogenes by ribozymes which are RNAs with site specific ligation activity [28].

Owing to treatment resistance and toxic side effects of cisplatin, implementation of combination therapy has gained much importance with regards to increased sensitivity of cisplatin towards cancer cells when combined with other drugs. The combination of other drugs with cisplatin may result in increased anticancer effect, there by result in reducing the dose of cisplatin and ultimately reduce toxic side effects. Hence, combination therapy may be adopted to evade cisplatin toxicity and resistance to cancer cells.

Combination therapy to overcome resistance and toxicity

Cisplatin and mitogen activated protein kinases (MAPKs)

MAPKs are a highly conserved family of structurally related serine/threonine protein kinases responsible for coordinating a variety of extracellular signaling pathways, regulating fundamental cellular processes involved in cell growth and survival [7, 9, 66]. There are three subfamilies in MAPK family, including ERK (member of Ras/MAPK), JNK and p38 kinases [10, 67, 68].

Although previous findings have identified cisplatin to result in ERK activation in several forms of cancer, whether this activation prevents or contributes to cisplatin-induced apoptotic effects remains greatly controversial [69–75]. Cisplatin-induced ERK activation precedes p53-mediated DNA damage response as ERK directly phosphorylates p53, resulting in upregulated expression of p21, 45kd-growth arrest and DNA damage (GADD45), and mouse double minute 2 homolog (Mdm2) [76]. As such, ERK activation may result in cell cycle arrest thereby supporting repair of DNA damage induced by cisplatin, via the tumor suppressor p53 (Fig. 2). Furthermore, p53 also directly affects the expression of downstream genes that regulates the sensitivity to apoptosis, activating transcription of Bax (promotes apoptosis) and repressing transcription of Bcl-2 (inhibits apoptosis) [28].

Furthermore, DNA damage results in the activation of JNK. The activation of JNK pathway results via both the cis and trans forms of cisplatin. DNA damage consequently leads to the stabilization and activation of p73, a pro-apoptotic protein related to p53, forming a complex with JNK, mediating apoptosis (Fig. 2) induced by cisplatin [77]. In addition, various other stress stimuli such as environmental stress are important mediators of apoptosis induced via cisplatin. This has been found to occur through the activation of p38 MAPK family. It was reported that cisplatin induces EGFR internalization mediated by p38 MAPK dependent phosphorylation of receptor [78]. Cisplatin induces stabilization of p18 (Hamlet), a protein regulated by p38 MAPK. This enhanced the ability of p53 to interact with and activate PUMA and NOXA, critical pro-apoptotic genes (Fig. 2). Therefore, p38 MAPK pathway plays a crucial part in the regulation of cisplatin ability to induce apoptosis [79]. Moreover, sustained activation of JNK/p38 MAPK pathways by cisplatin has resulted in inducing apoptosis in ovarian carcinoma cells [80]. Previous studies also showed that inhibition of either JNK or p38 kinase attenuated cisplatin induced apoptosis in cervical cancer cells, supporting the role of JNK and p38 in cisplatin response [81]. In contrast, foregoing study has reported that inhibition of p38 MAPK pathway resulted in upregulation of reactive oxygen species, mediating activation of JNK and thereby sensitizing human tumor cells to cisplatin induced apoptosis [82]. In addition, the same study has reported the use of mouse model for breast cancer to confirm that inhibition of p38 MAPK conjoins with cisplatin therapy to decrease tumor size and malignancy [82]. This is also in accordance with another study where p38 MAPK inhibition potentiates cisplatin induced apoptosis via glutathione depletion and drug transport alteration in epithelial renal tubule cell lines [83].

The MAPK cascade also includes upstream signal transducing molecules MEK (MAPK ERK kinase), Raf and Ras. Recent studies have reported the combination of MEK inhibitors along with cisplatin in in vitro and clinical trials [84, 85]. MEK inhibitor along with cisplatin resulted in enhanced apoptotic response in platinum sensitive ovarian carcinoma cells. Phase I/II clinical trials in biliary cancer patients observed significant activity of MEK inhibitor in combination with cisplatin in selected patients with prolonged and complete response to treatment.

Cisplatin and p90 ribosomal s6 kinase (RSK)

The RSK proteins are the downstream mediators of the Ras–ERK signal transduction pathway. ERK-mediated phosphorylation/activation of RSK plays an important role in regulation of protein translation, cell cycle progression and migration [10, 12, 13]. In addition, RSK proteins have been shown to induce proliferation and survival of cells by enhancing the expression of anti-apoptotic or pro-survival genes, and inhibiting pro-apoptotic genes [11]. A recent research study reported that RSK plays a stimulatory role on ethanol induced hepatocellular carcinoma progression by activating anti-apoptotic factor Bcl-2 and NHE1, known to regulate cell survival [86].

In humans, four RSK isoforms (RSK 1–4) and two structurally related homologs have been identified [87]. Although the RSK isoforms 1–3 are functional, there is a better understanding of RSK1 and RSK2, which display a higher similarity structurally, having a sequence identity of 73%. Furthermore, both isoforms have been associated with tumorigenesis in a wide range of human cancers [88]. More specifically, RSK1, reported to be more frequently activated in melanoma cancer cells, however, were found to be reduced in metastasized lung tissue compared to the primary tumor [8, 89]. Invasiveness and metastasis in head and neck squamous carcinoma cells has been shown to be promoted by RSK2. In addition, RSK2 has reported to contribute towards the survival of multiple myeloma cells [90, 91]. Breast and prostate cancer patients have also displayed higher expression of RSK1 and RSK2 compared to non-cancerous tissue [92, 93]. However, the functions of RSK4 does not resemble to the Ras–ERK pathway. RSK4 has been shown to be involved in p53 mediated cell growth arrest and in oncogene stimulated cellular senescence. The overexpression of RSK4 decreased breast cancer cell proliferation and promoted G0/G1 phase cell cycle arrest. RSK4 has also been reported to be over expressed in greater than 50% of primary malignant lung cancers. RSK3 and RSK4 facilitate resistance to PI3 kinase inhibitors in breast cancer [87]. However, recent research has shown that RSK4 is expressed at low levels in malignant ovarian tumors which correlate with advanced stages of the disease, and cisplatin increases the expression of RSK4 in SKOV3 and TOV112D ovarian cancer cell lines [94]. Therefore, the functions of RSK3 and RSK4 in cancer have not yet been clearly identified.

It is proposed that targeting anti-apoptotic signals associated with the promotion of cell survival may be a promising strategy to optimize the effectiveness of conventional chemotherapeutic agents. RSK mediated signaling involves the phosphorylation and inactivation of pro-apoptotic proteins and the activation of transcription factors and translational machinery that promote cell survival. RSK1 and RSK2 activate pro-survival proteins Bcl-2 and Bcl-xL by post translational phosphorylation and inactivation of the pro-apoptotic protein Bad, enhancing its ability to bind 14-3-3 proteins and preventing its heterodimerization with the pro-survival proteins. RSK1 and RSK2 also phosphorylate CREB leading to increased transcription of Bcl-2 and promote cell survival. Also, RSK1 directly inhibits caspase activity, promoting cell survival. RSK phosphorylates and inhibits GSK3β to promote stabilization of cyclin D1 resulting in cell cycle progression and cell-survival [87]. Previous study has reported that inhibition of RSK with a dihydropteridinone, BI-D1870, which is a potent RSK inhibitor, decreases cell migration and proliferation of A549 human lung adenocarcinoma cells, through phospho GSK 3β [14]. Other inhibitors of RSK include SL0101, LJH685, LJI308 and BIX 02565 which are ATP-competitive inhibitors of the N-terminal kinase domain of RSK, and FMK which is a covalent inhibitor of the C-terminal kinase domain of RSK [10]. Inhibition of RSK2 (ser227) with BI-D1870 induced apoptosis mediated cell death resulting in regressed myeloma cell proliferation [95]. Moreover, combination of BI-D1870 with mTOR inhibitor (everolimus) or histone deacetylase inhibitor (MS-275) or BH3-mimic inhibitor for BCL2/BCLXL (ABT-263), resulted in synergistic or additive anti-proliferative effects in myeloma cells suggesting that RSK2 (ser227) is a potential target in treating patients with multiple myeloma [95]. Furthermore, our own research has demonstrated the effect of combination treatment of BI-D1870 along with cisplatin on migration and apoptosis of A549 lung cancer cells. Our unpublished results indicate that BI-D1870 potentiates cisplatin induced apoptosis and inhibition of metastasis in non-small cell lung cancer. A recent study showed that inhibition of RSK with BI-D1870 overcame the drug resistance to inhibition of Sonic Hedgehog signaling pathway, which has been implicated in the pathogenesis of a variety of human cancers [87]. In a study on ovarian cancer cell line (A2780), treatment with cisplatin downregulated the expression of RSK2. Furthermore, silencing of RSK2 gave rise to improved cisplatin sensitivity [96]. These findings suggest that inhibition of RSK has the potential to sensitize tumors to anticancer agents.