Improvement in radiosensitivity by ERK inhibition
As aforementioned, radiation can induce rapid activation of ERK in various cancer cell types (50). A study has demonstrated that activated ERK protects cancer cells from the cytotoxic effects of radiation (51). ERKs promote cell survival in various ways, including via the inhibition of apoptosis, the induction of DNA damage repair and the arrest of the cell cycle.
Apoptosis, or programmed cell death type I, is an essential process leading to the removal of damaged cells without affecting normal cells, following DNA damage or during development (52). Apoptosis can be triggered by the caspase-mediated intrinsic signaling pathway, which is mainly regulated by the B-cell lymphoma 2 (Bcl-2) family of intracellular proteins, or by an extrinsic signaling pathway, which is closely regulated by the tumor necrosis factor (TNF) receptor family (53,54). Deregulation of apoptosis is associated with uncontrolled cell proliferation, cell growth, progression of cancer and cancer resistance to drug therapies (55,56). Therefore, apoptosis deregulation is considered a hallmark of cancer (55). Two types of proteins determine the cellular outcome: Pro-apoptotic proteins, such as Bcl-10, Bak, Bid, BAG, Bax, Blk, Bad and Bim, and anti-apoptotic proteins, such as Bcl-2, Bcl-x, Bcl-XS, Bcl-xl, Bcl-w, IAP and Mcl-1 (56). ERK activation induced by IR can activate various substrates, which transmit the signaling of ERK to apoptosis-associated proteins; common substrates include transcription factors, such as nuclear factor erythroid 2-related factor 2 (NRF2), cyclic AMP-responsive element binding protein (CREB) and CAAT/enhancer binding protein β (C/EBP-β) (57,58). Chen et al (57) observed that radiation-induced ERK1/2 phosphorylation increased NRF2 expression in osteosarcoma U-2 cells; activation of NRF2 served a radioprotective role by stimulating Bcl-2 and p65 expression, while inhibiting Bax and p53 expression. Similarly, ERK1/2 can induce Bcl-xl, Mcl-1 and c-FLIPs expression via CREB and C/EBP-β activated by ERK1/2 (38). Additionally, ERK is able to activate the NFγB1 dimer, a crucial regulator of anti-apoptotic genes, including genes encoding inhibitors of apoptotic proteins and members of the Bcl-2 family (59). Furthermore, ERK can directly stimulate Bid and Bim (38). Therefore, ERK1/2 is associated with apoptosis and can protect against cell death mainly by increasing or decreasing levels of anti- and pro-apoptotic proteins, respectively.
DNA damage repair is essential in the cancer cell response to IR and includes at least five main processes: Base excision repair, nucleotide excision repair, mismatch repair, homologous recombination (HR) repair and non-homologous end joining (NHEJ) (60). HR and NHEJ, the major repair pathways for DNA DSBs and closely associated with ERK (61), are mainly regulated by PI3K-like kinases, including ataxia telangiectasia mutated (ATM), RAD3-related protein (ATR) and DNA-dependent protein kinase (DNA-PK) (62). Additionally, DNA DSB repair is modulated directly or indirectly by other means, such as EGFR and the ERK axis (63). ERKs can directly activate ATM or DNA-PK, which mainly participate in NHEJ-mediated DSB repair (4). Furthermore, ERK can activate ATR followed by cell cycle arrest (22). Additionally, activation of ERK induced by IR has been associated with increased levels of transcriptional proteins (such as ERCC1 and XRCC1) involved in DNA repair in DU145 and LNCaP prostate carcinoma (64,65). However, in one study, radiation-induced ERK activation was affected by the extent or scope of DSB: Low-level DSBs (equivalent to 2 Gy) resulted in ERK activation, while high-level DSBs (>2 Gy) led to phosphatase-mediated ERK dephosphorylation and subsequent suppression of the ERK signaling pathway (4). Therefore, activation of ATM or ATR by activated ERK may depend on the radiation dose. Overall, ERK seems to be associated with DNA repair to protect against cell death.
Cell cycle arrest, which is governed by cell cycle checkpoint-associated proteins, such as cell cycle proteins (cyclins) and cyclin-dependent kinases, can maintain gene stability by blocking cell cycle progression and initiating processes to repair the detected damage (66). Some studies have demonstrated that ERK1/2 pathway activation following IR or DNA damage is critical for the activation of cell cycle checkpoints in response to radiation cytotoxicity; these studies confirmed that phosphorylation of ERK1/2 is a prerequisite for inducing ATR expression, which can activate Wee1 and checkpoint kinase 1 as key regulators of the G2/M checkpoint (67,68). Additionally, one study indicated that expression of basal breast cancer type 1 susceptibility protein (BRCA1) tumor suppressor is necessary for IR-induced activation of ERK, followed by G2/M arrest, in MCF-7 cells (69). Therefore, ERK is associated with key cell cycle checkpoint proteins to induce cell cycle arrest, and serves a protective role in response to radiation cytotoxicity.
Previous studies have demonstrated that ERK signaling serves a radioprotective role in cancer therapy and contributes to radioresistance (9,29,70). Therefore, inhibition of the ERK signaling pathway may provide a valuable approach to increase the radiosensitivity of cancer cells in response to IR. Various inhibitors for suppressing ERK signaling have been tested in preclinical and clinical investigations, including RAF inhibitors (vemurafenib, dabrafenib, LGX818, TAK-632, MLN2480 and PLX-4720), MEK inhibitors (trametinib, cobimetinib, MEK162, AZD6244, RO5126766, GDC-0623 and PD0325901) and ERK inhibitors (SCH772984, VTX11e and GDC-0994) (10,11,71,72). Radiation in combination with RAF and MEK inhibitors has been widely tested in vitro and in vivo for multiple types of cancer cells and various biological mechanisms (73). Estrada-Bernal et al (74) demonstrated that GSK212, a MEK1/2 inhibitor, downregulates several intermediates of DSB repair signaling, including BRCA1, RAD51, DNA-PK and PPM2, in irradiated pancreatic cancer cells, and suppresses DSB damage repair, particularly through HR repair pathways. Similar results were obtained by Marampon et al (75) using another MEK1/2 inhibitor, U0126, which reduced DNA-PK expression induced by IR. Furthermore, radiosensitization induced by a MEK inhibitor, AZD6244, was associated with a decreased cell cycle checkpoint response and increased mitotic catastrophe compared with no inhibitor present; in these in vitro and in vivo experiments, an apparent growth delay in xenografts of A549 cancer cells was observed after AZD6244 was combined with irradiation, compared with treatment by irradiation alone. Notably, the DNA repair pathway did not differ between the combined model and each treatment alone, which may be attributable to differences in molecular structure. A study observed that ERK inhibitors sensitized cancer cells to irradiation by downregulating specific molecules, such as transforming growth factor-α and TNF-α converting enzyme, which can stimulate a radioresistance mechanism by activating EGFR after inhibiting mutant RAS (76). Therefore, ERK inhibitors may increase cell death by suppressing phosphorylation of EGFR signaling pathways. Other potential mechanisms, such as ERK inhibition, can disrupt the production of c-Myc induced by IR, which can promote the onset, progression and resistance to targeted therapy in numerous types of cancer, such as prostate cancer (6,77). Active ERK-induced apoptotic effects have been confirmed by promoting pro-apoptotic and inhibiting anti-apoptotic proteins (59). The aforementioned data suggest that inhibition of ERK signaling may be an effective treatment for some types of cancer cells.
The effects of inhibitors of ERK signaling on tumor suppression are being evaluated in various clinical trial phases; however, the observed poor outcomes are the result of acquired resistance, clinical side effects, varying functional times, intensity or other unclear reasons, which are limiting their value for clinical application (78–80). The main mechanisms of acquired resistance include: i) NRAS or KRAS mutations, amplification of BRAF V600E, mutations in MEK1/2 and loss of CDKN2A, which lead to BRAF inhibitor resistance; ii) MEK mutations or BRAF amplification, which result in MEK inhibitor resistance; iii) ERK mutations, which contribute to ERK inhibitor resistance; iv) other abnormally activated or elevated levels of molecules, such as PI3K/AKT, CCND1, receptor tyrosine kinase and CRAF128; v) a decreased dependency of tumor cells on the ERK signaling pathway for growth; and vi) rebound of MEK/ERK after inhibition treatment or compensatory mechanisms (11,18,81).
Numerous studies have revealed less satisfactory outcomes regarding ERK inhibition (11,12). Therefore, the opposing functions of active ERK must be considered, particularly when evaluating whether radiosensitivity may be improved by inducing ERK activation.
Activation of ERK: A potential strategy for radiosensitization
Some studies have suggested that activation of ERK by IR or other compounds may lead to cell death; active ERK can have a pro-death or growth-arrest role in cancer cells (15,82). Dual effects are the result of the numerous mechanisms upstream and downstream of ERK. However, the association between the pro-death effect of active ERK and radiosensitivity has not been systematically evaluated in cancer cells.
Studies have identified various stimuli that can further induce cancer cell death by activating ERK, including antitumor agents, such as taxol (83), carboplatin (48), etoposide (84), doxorubicin and cisplatin (85), elements, such as cadmium (86,87) and benzo(a)pyrene (88), irradiation (89,90), naturally derived products, such as chelerythrine (91) and piperlongumine (92), and others (82,93). Additionally, constitutively activated ERK mediates cell death. In two diffuse large B-cell lymphoma (DLBCL) cell lines, CD40-sensitive DLBCL cells were induced to undergo apoptosis by CD40 ligand (CD40L) only when ERK was constitutively activated, and this effect disappeared when the MEK inhibitor U0126 inhibited ERK phosphorylation; by contrast, CD40-resistant DLBCL cells exhibited no response to CD40L due to a lack of constitutively activated ERK (82). Three main pro-death or anti-proliferative mechanisms were involved in this process, including apoptosis-induced cell death, autophagic programmed cell death and senescence (15,94,95).
As aforementioned, both constitutive and stimuli-induced ERK signaling contribute to apoptosis and are influenced by numerous factors, some of which are closely associated with IR or radiosensitivity. Lee et al (47) demonstrated that overexpression of protein kinase C δ (PKCδ), a PKC isoform, contributed to the expression of phosphorylated ERK; the PKCδ-ERK signaling pathway further enhanced radiation-induced apoptosis and radiosensitivity by abrogating radiation-induced G2/M arrest. Another isoform of PKC, PKCε, also led to radiation-induced cell death by mediating ERK activation; this pro-death effect was inhibited when NIH3T3 cells were pretreated with the MEK inhibitor PD98059 (69,89). Watanabe et al (90) demonstrated that PD98059 significantly inhibited radiation-induced apoptosis, further leading to radioresistance, indicating that active ERK may serve a radiosensitizing role in rat cells. In terms of ERK substrates, several studies have demonstrated that activated ERK in numerous cancer cells can phosphorylate p53, an essential tumor suppressor that serves a pivotal role in protecting genome integrity and mediating cell death (96–98), and is key to the radiation response in tumor cells. Functional p53 regulates irradiated cancer cell death by inducing apoptosis or senescence, and by inhibiting autophagy-associated cell survival (30,49,99). Pseudo-ginsenoside-Rh2, which is a derivative of ginsenoside Rh2, has been shown to suppress cell growth and induce intrinsic apoptotic pathways through activation of the Ras/Raf/ERK/p53 signaling pathway, subsequently upregulating Bax expression and downregulating Bcl-2 and Bcl-xl expression (100). Furthermore, excessive activation of the Ras/Raf/ERK/p53-p21 signaling pathway was shown to induce apoptosis and G2/M arrest in human lung carcinoma cells (101). A study demonstrated that activation of ERK/p53/Beclin-1 could mediated autophagic cell death in A549 cells (102). Therefore, p53 upregulation seems to be an essential mechanism of ERK-induced cell death (Fig. 2). However, other studies have suggested that ERK induces cell death independently of p53 such as cPLA2-FasL and DAPK-TNF pathway (83,103).
In addition, there are a number of other factors for which the intensity and localization of ERK activation may be key in the choice between cell survival or cell death (104). Tang et al (84) demonstrated that etoposide and IR resulted in DNA damage that activated ERK with the same intensity; however, low-intensity DNA damage resulted in cell cycle arrest, while high-intensity DNA damage caused apoptosis. Inactive ERK localizes in the cytoplasm, while activated ERK1/2 typically exerts its role by entering the nucleus and phosphorylating transcription factors (105). However, some studies have suggested that active ERK1/2 can access specific substrates and affect cell conditions by translocating to other organelles, such as the mitochondria (106), the endoplasmic reticulum (107) and various membranes (108). Studies have demonstrated that nuclear ERK mainly improves cell proliferation or oncogenic transformation and migration in various types of cancer either by inducing oncogenic signals, such as c-Myc or c-Fos, or by inhibiting tumor suppressors such as Tob or Foxo3a (109–112). However, stimulation of mitochondrial ERK is involved in both cell survival and apoptosis (105). For example, Cook et al (106) demonstrated that activation of the mitochondrial ERK1/2 signaling pathway promoted mitochondrial fission or fragmentation, with the latter being involved in the onset of cell apoptosis. A previous review stated that the specific mechanisms of the aforementioned translocation are mediated by a number of anchoring and scaffold proteins (105).
Overall, the aforementioned data suggest that activating, rather than inhibiting, ERK may increase cancer cell death. Therefore, an activator of ERK, honokiol, has been tested in vitro and in vivo. Honokiol exhibits the potential to treat cancer by inhibiting cell growth and migration by inducing ERK-dependent apoptosis and autophagy (113,114). Therefore, ERK-induced cell death may be a potential therapeutic strategy that requires further evaluation.