Apoptosis

Apoptosis is a form of programmed cell death characterised by specific morphological changes (Ref. Reference Kerr, Wyllie and Currie21), which consists of two major subtypes, namely the extrinsic and intrinsic apoptotic pathways (Fig. 3). Extrinsic apoptosis is mediated by membrane receptors, especially by death receptors (e.g. Fas cell surface death receptor and tumour necrosis factor (TNF) receptor superfamily member), and is driven mostly by the initiator caspases 8 and 10 (Ref. Reference Schulze-Osthoff22). Initiator caspase 9 can also trigger extrinsic apoptosis together with unc-5 netrin receptor B and DCC netrin 1 receptor (Ref. Reference Bredesen, Mehlen and Rabizadeh23), although this is mostly involved in the activation of the intrinsic apoptotic pathway. Intrinsic apoptosis starts with mitochondrial outer membrane permeabilisation which is controlled via a fine balance of BCL2 family pro- and anti-apoptotic members, including BCL2-associated X, apoptosis regulator (BAX), BCL2 antagonist/killer 1 (BAK1) and BCL2 and BCL2-like 1 (BCL2L1) (Refs Reference Singh, Letai and Sarosiek24, Reference Czabotar25). When the pro-apoptotic signal overcomes the anti-apoptotic one, mitochondrial proteins are released into the cytoplasm (e.g. cytochrome C and diablo IAP-binding mitochondrial protein) and this triggers initiator caspase 9 activation (Ref. Reference Chipuk, Bouchier-Hayes and Green26). Both the intrinsic and extrinsic pathways of apoptosis proceed with the activation of effector caspases (caspases 3, 6 and 7), which in turn catalyse the specific cleavage of many key cellular proteins. Other members of the cysteine-dependent aspartate-specific protease family are caspase 2 (initiator caspase), caspases 1, 4, 5, 11 and 12 (inflammatory caspases) and caspase 14 (keratinisation-relevant caspase). In terms of morphological features, apoptotic cells show chromatin condensation which progresses into nuclear fragmentation as the apoptotic process proceeds, and this ends with the formation of apoptotic bodies and phagocytosis by the surrounding cells (Ref. Reference Kerr, Wyllie and Currie21).

Fig. 3. The extrinsic and intrinsic pathways of apoptosis. The extrinsic death receptor pathway is activated by death receptor ligands, including FasL, TNF-α or TRAIL, which in turn activates caspase 8 and downstream executing caspases. The intrinsic death receptor pathway is initiated by several intracellular stresses, leading to activation of Bax and Bak on the mitochondrial membrane and which result in the release of cytochrome c from the mitochondria. Cytoplasmic cytochrome c activates caspase 9 and downstream executing caspases.

Necrosis

Necrosis is usually induced by several physical or chemical stress factors, including ischemia and hypoxia. The main event in necrosis is mitochondrial inner membrane depolarisation and outer mitochondrial membrane rupture, due mostly as a consequence of an increase in Ca²⁺ ions, ATP depletion and reactive oxygen species production (Ref. Reference Vanlangenakker27). However, other metabolic changes have been observed in cells undergoing necrotic death (Ref. Reference Brown28). Necrotic cell death can also be induced when ligands bind to specific receptors, such as TNF receptor 1, Fas and TRAIL receptor, although these activation pathways are equally shared within apoptosis (Refs Reference Peter and Krammer29, Reference Wajant, Pfizenmaier and Scheurich30). Necrosis is also induced by biological stress triggers such as external pathogens, which are recognised by pathogen recognition receptor family members, including the membrane-associated Toll-like receptors, the cytosolic NOD-like receptors and RIG-I-like receptors (Refs Reference Gay and Gangloff31–Reference Loo and Gale33). At a macroscopic level, necrosis stimulates the cell membrane to become permeable early in the process, followed by leakage of the cellular contents. In the case of autophagic-like necrosis, numerous vacuoles are observed in the cytoplasm, while dilation of organelles and empty spaces is detectable if the cell undergoes non-lysosomal-type necrosis (Ref. Reference Ziegler and Groscurth34).

Autophagy

Autophagy is a highly regulated mechanism by which the cell removes unnecessary or dysfunctional components by self-degradation and recycling within the cell. Autophagy can be categorised into three forms: macroautophagy (or simply autophagy), microautophagy and chaperone-mediated autophagy (Fig. 4). Macroautophagy requires an intermediate single-membrane vesicle called a phagophore that engulfs the cytoplasm containing the components to be degraded, and rounds up becoming a double-membrane autophagosome. This will then fuse with an endosome or lysosome and releases its contents into the lytic organelle for degradation. Microautophagy, on the other hand, does not have any intermediates and the cellular content to be removed is directly engulfed by an endosome or lysosome (Ref. Reference Schuck35). Chaperone-mediated autophagy requires firstly the recognition of a specific motif in the protein to be degraded by heat shock cognate 71 kDa protein (hsc70), which will then bind the cytosolic tail of lysosome-associated membrane protein type 2A on the lysosomal membrane and transport the substrate into the lysosomal lumen where it will be rapidly degraded (Ref. Reference Tekirdag and Cuervo36). The proteins involved in vesicle formation can be grouped together as the autophagy-related (ATG) proteins, which collectively provide a very dedicated and fine machinery (Ref. Reference Yu, Chen and Tooze37). Autophagic responses are usually triggered by nutrient deprivation, but also by organelle damage or by intracellular pathogens (Ref. Reference Mizushima and Komatsu38). Since autophagy may act as a tumour suppressor, an impaired mechanism could lead to the accumulation of toxic proteins and organelles, such as dysfunctional mitochondria, thus promoting oxidative stress, accumulation of DNA lesions and genomic instability, ultimately leading to promotion of cancer transformation (Ref. Reference Gozuacik and Kimchi39). Recently, it has been suggested that basic autophagy can actually maintain the survival of cancer cells in their unique environment (Ref. Reference Kang40).

Fig. 4. The mechanisms of autophagy. Schematic representation of the three main autophagy pathways: macro-, micro- and chaperone-mediated autophagy. Macroautophagy sequesters cytosolic cargo inside a phagophore formed by specific ATG proteins and lipids. The membrane then seals into an autophagosome and fuses with lysosomes causing the degradation of the trapped cargo. Microautophagy entraps cytosolic cargo in small vesicles formed by invagination of the lysosomal membrane. Chaperone-mediated autophagy involves the selective degradation of KFRQ-like motif-bearing proteins delivered to the lysosomes via chaperone HSC70 and their internalisation in lysosomes via the receptor lysosome-associated membrane protein type 2A (LAMP2A).

Senescence

The term ‘replicative cellular senescence’ indicates a process in which cells are alive but are unable to undergo further cell division and therefore are in a permanent state of growth arrest (Ref. Reference Ben-Porath and Weinberg41). Senescence can be triggered by a variety of factors and is usually associated with increased levels of p53, p21^WAF–1 and/or p16^INK4A, but telomere-associated DNA damage foci are also often present (Ref. Reference Kuilman42). The molecular mechanisms involved in senescence pathway activation and execution have been extensively studied and reviewed (Refs Reference Ben-Porath and Weinberg41, Reference Chakradeo, Elmore and Gewirtz43). Assessment of senescent cells can be easily performed with histochemical staining of β-galactosidase (Refs Reference Kurz44, Reference Yang and Hu45), although other markers of senescence that have been identified include Cathepsin D and Dec-1 (Refs Reference Byun46, Reference Kim47). Morphological changes in senescent cells include increased granularity and cytoplasmic vacuolar content, followed by cell flattening. Senescence has always been considered an irreversible process, due to the major metabolic modifications the cell undergoes together with genetic and structural alterations, although several studies have highlighted the possibility of cells escaping senescence. In fact, in the case of senescence induced by chemotherapy or RT, several studies have reported that cells were able to re-enter the cell cycle due to overexpression of the cyclin-dependent kinase, Cdc2/Cdk1 or its downstream target survivin (Refs Reference Roberson48–Reference La Porta, Zapperi and Sethna50). Considering that most tumours have inactive p53 and/or p16INK4A/Rb signalling pathways, escaping senescence should be considered as a possibility.

Mitotic catastrophe

The term ‘mitotic catastrophe’, or the more appropriate phrase ‘mitotic death’, indicates cell death induced by aberrant mitosis (Ref. Reference Galluzzi17). Cells undergoing mitotic catastrophe show unique morphologically defined features, such as multinucleation and micronucleation, mainly due to the chromosomal mis-segregation characteristic of this process (Ref. Reference Vitale16). Mitotic death can be triggered by either exogenous or endogenous sources, which ultimately cause several cell dysfunctions, such as altered DNA replication and chromosome segregation, and interference with microtubular dynamics (Refs Reference Dominguez-Brauer51–Reference Denisenko53). The molecular mechanisms involved in mitotic catastrophe are still under investigation, although p53 seems to play a role in the trigger signal (Ref. Reference Vitale16) and to activate the transduction cascade via caspase 2, which in turn initiates a variant of intrinsic apoptosis regulated by members of the BCL2 protein family (Refs Reference Dawar54, Reference Lopez-Garcia55). However, many cancer cell types lack functional p53 and in those cases it appears that cells showing massive chromosomal aberration are driven preferentially into necrosis (Ref. Reference Surova and Zhivotovsky56). It is interesting to note that cell death is not the only possible fate aberrant mitotic cells can face, as it has been observed that cells escaping the mitotic block can instead enter p53-mediated or hippo-mediated cellular senescence (Refs Reference Ganem57–Reference Meitinger60), or survive as polyploid and aneuploid cells and initiate neoplastic transformation and progression (Ref. Reference Vitale16). However, a recent study suggests that cells escaping mitotic catastrophe stimulate the immunological response which in turn will trigger cell degradation (Ref. Reference Mackenzie61), providing an efficient mechanism for the control of tissue homeostasis.

Cell death mechanisms after low LET exposure

As mentioned above, tumour cells post-IR can undergo apoptosis, although recent studies have implicated a reduced contribution of this particular pathway to the total amount of cell death to a relatively low level (Refs Reference Rodel67, Reference Gewirtz68). Apoptosis detection in several tumour cell lines, including breast cancer, non-small-cell lung cancer and colorectal cancer, has been reported to never exceed 30% of the total even at significantly high doses of radiation (Ref. Reference Chakradeo69). HNSCC cell lines appear to show no difference in the levels of IR-induced apoptosis, with the vast majority of the studies agreeing that low-LET radiation (e.g. X-rays and γ-rays) does not cause significant apoptosis activation (summarised in Table 1). In fact, thyroid cancer cells show no apoptotic response after a relatively low dose of 3 Gy (Ref. Reference Suzuki70), and only a modest increase after 20 Gy treatment (Ref. Reference Kim71), whilst laryngeal squamous carcinoma cells appear to reach an increase in 20% of apoptotic cells after a fractionated 10 Gy dose (Ref. Reference Wang72). The HNSCC cell lines UM-SCC1, UM-SCC6 (both HPV-negative) and UPCI-SCC-154 (HPV-positive) showed no difference in caspase 3 activation and Annexin V detection at 2 and 4 Gy X-ray exposure (Refs Reference Kahler73, Reference Kashin74). Similarly in nasopharyngeal carcinoma cell lines, no difference in apoptosis was observed after 8 Gy (Ref. Reference Zhan and Ni75), although in contrast another study showed a 10-fold increase in the apoptotic response after 10 Gy (Ref. Reference Nie76).

Table 1. Biological effects of low-LET radiation on head and neck cancer cells

^a X-ray radiation treatment unless differently stated.

Necrosis pathway activation has mostly been considered more as a side-effect of RT in surrounding healthy tissue, rather than a cell death mechanism for cancer cells. Patients undergoing RT for HNSCC can experience significant side-effects, including osteoradionecrosis and oral cavity necrosis, which may require surgical intervention (Ref. Reference Zehr and Cooper87). The mechanism underlying this specific necrotic transformation is not fully understood, although fibrosis may play a major role. This is due to the fact that RT increases the levels of reactive oxygen species-mediated cytokines, such as TNF-α, transforming growth factor-β1 and connective tissue growth factor, resulting in unregulated fibroblastic activation (Ref. Reference Dambrain88). Human and mouse leukaemia cells have been shown to undergo necrosis after 300 and 9 Gy X-ray exposure, respectively (Refs Reference Nakano and Shinohara89, Reference Akagi, Ito and Sawada90), suggesting the radiosensitive nature of the murine cells. HNSCC cells appear to undergo necrosis after X-ray radiation exposure at different degrees depending on cell type and HPV status (summarised in Table 1). In fact 48 h after irradiation, HPV-negative SqCC/Y1 cells showed a significantly higher percentage of necrotic cells compared to the unirradiated controls, but this was not observed in another HPV-negative cell line (HN5) or in two HPV-positive cell lines (UPCI-SCC-154 and UMSCC-47) tested (Ref. Reference Wang77). One explanation, at least in the HPV-positive HNSCC cell lines, could be provided by the fact that the HPV oncoprotein E7 inhibits necrosis activation, thus making these cell lines less prone to this mechanism of cell death (Ref. Reference Shankar91).

Since autophagy is mostly associated with tumour suppression, increases in ATG proteins are to be expected following RT, and have been observed in HNSCC (summarised in Table 1). The ratio of LC3II/LC3I (an autophagic marker) has been shown to increase significantly in nasopharyngeal carcinoma cells after exposure to 4 Gy X-rays (Ref. Reference Liu78), and protein level analysis showed that autophagic signalling proteins including Beclin1, Atg5, Atg7 and LC3B were upregulated in a time- and dose-dependent manner post-irradiation (Ref. Reference Jing92). However, the same treatment did not appear to trigger any LC3B activation in SQ20B carcinoma cells (Ref. Reference Cerniglia79), suggesting a role for a differential gene expression profile in contributing to autophagy specific for each cell line. Exposure to 6 Gy of low-LET X-rays radiation caused a 2–5-fold increase in several ATG proteins in Cal-33 carcinoma cells, including LC3B, p62, Atg4A and Atg4B (Ref. Reference Deville80). However, although autophagy directly contributes to death in stressed cells (Ref. Reference Ryter, Mizumura and Choi93), several studies have actually suggested a protective role for autophagy, which actually helps cancer cells to survive by reducing cell damage (Ref. Reference Mathew, Karantza-Wadsworth and White94). Autophagy is frequently activated in radioresistant cancer cells (Ref. Reference Han95) and the pathway shares upstream mediators with apoptosis, indicating a clear cross-talk between the two mechanisms and a fine balance between cell death and cell survival in response to IR. It has been proposed, in fact, that low-energy X-rays trigger the NF-κB pathway and induce Beclin1 gene expression, which consequently activates autophagy and confers radioresistance in HNSCC cancer cells (Ref. Reference Shu96). Moreover, apoptosis can be inhibited by autophagy proteins, such as Beclin1, which can degrade caspase 8 and interfere with the activation of Bid (Ref. Reference Nikoletopoulou97), reducing cell death levels after irradiation (Ref. Reference Shu96).

Senescent cells induced by RT usually trigger inflammation, which in turn leads to the targeted removal of cancer cells (Ref. Reference Chang98), making senescence an outcome at the initial treatment stages. In contrast, some studies suggest the possibility of senescence being a protective mechanism for cancer cells, causing increased aggressiveness and metastasis (Refs Reference Milanovic99, Reference Kim100). Relatively radiosensitive SCC61 HNSCC cells were demonstrated to accumulate 76% of senescent cells, while radioresistant SQ20B cells only displayed 18% of cells positive for senescence markers after 10 Gy γ-ray exposure (Ref. Reference Ricco81) (summarised in Table 1). X-ray (4 Gy) treatment significantly increased the percentages of senescent cells at 4 and 6 days post-IR in two HPV-negative HNSCC cell lines (SqCC/Y1 and HN5) and two HPV-positive cell lines (UPCI-SCC-154 and UMSCC-47), although the absolute percentage only ranged from 5 to 18% (Ref. Reference Wang77). In contrast, Detroit562 HNSCC cells (HPV-negative) showed no increase in the levels of senescence markers, such as p21 and β-galactosidase, after 2 Gy X-rays (Ref. Reference Frederick82). The potentially higher level of senescent cells present following irradiation of HPV-positive cells can be partially explained by the ability of the E2 oncoprotein to induce overexpression of p21 and downregulation of E7, resulting in increased levels of hypophosphorylated pRb protein and ultimately to G1 cell cycle arrest (Ref. Reference Wells101). The nasopharyngeal carcinoma cell line CNE2 showed around 56 and 79% IR-induced cellular senescence 72 h after 6 and 10 Gy X-ray exposure, respectively (Ref. Reference Yu83). Since CNE2 cells are p16-deficient, only p53 and p21 protein levels were elevated together with the anti-apoptotic protein Bcl-xL, suggesting senescence was preferred over apoptosis as the cell death mechanism. Conversely, it has been shown that only 5% of p53-deficient HNSCC cancer cells undergo senescence after 4 Gy X-rays radiation treatment, whilst p53-wild type containing cells reach up to 60% senescence-associated β-galactosidase-positive cells (Ref. Reference Fitzgerald102). This confirms the prominent role of p53 in driving senescence compared to p16. This confirmed previous observations in 38 different HNSCC cell lines where p53 mutation significantly decreased IR-induced senescence and conferred radioresistance (Ref. Reference Skinner103). Furthermore, the importance of the p53–p21 interaction for the initiation of the senescence pathway has been highlighted (Refs Reference Fitzgerald102, Reference Skinner103).

HNSCC cells exposed to 1 Gy γ-radiation have been shown not to display any significant induction of mitotic catastrophe (Ref. Reference Gottgens104), whilst a higher dose (6 Gy) of X-rays caused 20% of FaDu cells to undergo this process (Ref. Reference Yun84) (summarised in Table 1). The increase in mitotic catastrophe induction appears to be p53-independent, in line with previously reported evidence for HCT116 colorectal cancer cell lines after 4 Gy X-ray treatment (Ref. Reference Hafsi105). Oesophageal squamous cell carcinoma cell lines have demonstrated a modest increase in mitotic catastrophe markers after 6 Gy X-ray irradiation (Ref. Reference Cheng85), while nasopharyngeal carcinoma cells displayed around 50% of cells undergoing mitotic catastrophe after 20 Gy of X-ray treatment (Ref. Reference Lv86). Whilst the dose used is different, this could suggest that triggering of mitotic catastrophe as a cell death mechanism could be tumour cell-dependent. Mitotic catastrophe was observed to be the dominant mechanism of cell death in HPV-positive and HPV-negative HNSCC cell lines after a single 4 Gy X-ray radiation exposure, with no major difference between 4 and 72 h timepoint (Ref. Reference Wang77). Moreover, both types of cell lines tested responded in a similar manner, suggesting that HPV infection has no dramatic impact on the degree of mitotic catastrophe induction.

In summary, evidence suggests that low-LET radiation exposure may trigger different types of HNSCC cell death mechanism, and that the cells undergo a specific pathway depending on the radiation dose but also the cellular genetic profile.

Cell death mechanisms after high LET exposure

Mechanistic modelling has suggested that high-LET radiation, such as α-particles and heavy ions, can cause up to 90% of CDD within the total amount of DNA lesions induced, compared to ~30% in the case of low-LET radiation exposure (Refs Reference Nikjoo106, Reference Watanabe, Rahmanian and Nikjoo107). CDD has been clearly demonstrated to be a challenge to the DNA repair machinery and can persist in cells and tissues several (6–24) hours after IR, while simple SSBs and DSBs resolve in less than 30 min and 2 h, respectively (Refs Reference Carter64, Reference Magnander108–Reference Nickson111). High-LET radiation can therefore kill cancer cells more efficiently compared to low LET treatments. For example, and as reported for SQ20B laryngeal squamous cells, exposure to 4 Gy low-LET X-rays produced 56% cell survival whereas high-LET carbon ion (LET = 184 keV/μm) at the same dose yielded only 9% of cells surviving (Ref. Reference Bertrand112) (Table 2). A reduction in cell survival from 55 to 15% was also observed in UMSCC74A after 4 Gy high-LET proton treatment (LET = 12 keV/μm) compared to 4 Gy low-LET proton exposure (LET = 1 keV/μm), while UMSCC6 cells displayed a less marked difference (35% cell survival for low-LET versus 20% for high-LET) (Refs Reference Carter64, Reference Nickson113). This demonstrates the differences in inherent radioresistance of the different HNSCC cells to both low and high-LET radiation. Nevertheless, CDD formation is likely the major contributor to this observed reduced survival in response to high-LET radiation thereby triggering apoptosis or mitotic catastrophe, although other mechanisms in cell death pathway activation are likely to be involved once the cell fails to resolve the damage. For example, effects on centrosome biology in HNSCC cell lines have recently been shown in response to high-LET protons (Ref. Reference Nickson113).

Table 2. Biological effects of high-LET radiation on head and neck cancer cells

Interestingly, the mechanisms of cell death in response to high-LET radiation in HNSCC cells have not been studied extensively. Human tongue carcinoma cells have been shown to display less than a 5% increase in apoptotic marker activation after 5 Gy high-LET carbon ion exposure (LET = 70 keV/μm) compared to X-ray radiation (Ref. Reference Asakawa114), although this was not apparent when the dose was reduced to 1 Gy (Ref. Reference Takahashi115) (Table 2). Apoptosis activation has also been reported in a study on radiosensitive human oesophageal carcinoma cells, where caspase 3 activation was 1.8-fold higher after heavy ion irradiation versus X-rays, while only marginal differences were observed in their radioresistant cell counterparts (Ref. Reference Hartfiel116). This suggests that the cell-killing effect is cell line and radiation quality-dependent, and that perhaps other types of cell death may play a more prominent role. This confirms previous observations in radiosensitive laryngeal squamous cancer cells and their appropriate radioresistant cell lines (Ref. Reference Maalouf117). Considering the significant increased therapeutic use of PBT, but also heavy ion radiotherapy, further and more extensive studies are needed to fully elucidate the cell death mechanisms activated in specific HNSCC cell types in response to high-LET radiation exposure.

Therapeutic strategies

Targeting key proteins involved in cell death mechanisms is a feasible approach in increasing HNSCC radiosensitivity, which can lead to more effective treatment of the tumour. Some targets involved in cell death pathway activation and/or execution have been already identified and explored in vitro. In most cases, tumour cells highly express anti-apoptotic proteins, therefore their inhibition could potentially increase cellular radiosensitivity. Survivin is a protein inhibitor of the terminal effector caspases and is highly expressed in several types of cancer, including head and neck malignancies (Refs Reference Ambrosini, Adida and Altieri118, Reference Frassanito119). Efficient downregulation of survivin has been extensively studied with promising results in terms of enhancing tumour sensitivity to various therapeutic interventions (reviewed in (Ref. Reference Santarelli120)). Livin, another member of the apoptosis inhibitor protein family, has been found to be associated with tumour progression and poor prognosis in various human cancers. Its inhibition has been shown to suppress tumour cell invasion and enhance cell apoptosis, with elevated expression levels of cleaved caspases 3 and 7 and cleaved PARP in anaplastic thyroid cancer (Ref. Reference Kim71) and in laryngohypopharyngeal cancer models (Ref. Reference Yoon121). Other anti-apoptotic proteins, including Bcl-2 and vitronectin, have been investigated in several HNSCC subtypes, with their inhibition significantly increasing apoptosis in response to X-rays (Refs Reference Guy122–Reference Jiang124) or γ-radiation (Ref. Reference Zerp125). A phase I/II clinical trial initiated to investigate mitochondria-derived activator of caspases as a mimetic therapy in patients with previously untreated stage III/IV HNSCC in combination with cisplatin and radiation is currently ongoing (ClinicalTrials.gov identifier NCT02022098).

Post-radiotherapy, necrosis usually occurs in the oral cavity, maxilla, mandible and salivary glands more than the tumour itself, and the aim therefore is to prevent this to improve patient quality of life. Although several therapies have been reported, there is not a universally accepted approach to tackle necrosis and the treatment options are variable. Surgical treatments are still of preference, but recently some pharmacological options have been investigated, including treatments with antioxidants or biological molecules, such as basic fibroblast growth factor or bone morphogenetic protein-1 (reviewed in (Refs Reference Pitak-Arnnop126, Reference Madrid, Abarca and Bouferrache127)). Nevertheless, inducing necrosis in HNSCC cells is a possibility that has been partially explored. The monoclonal anti-IGF-1R antibody A12 in combination with γ-rays has been shown to dramatically induce necrotic death in FaDu-derived xenografts compared to unirradiated controls (Ref. Reference Riesterer128), while the second mitochondria-derived activator of caspase mimetic birinapant has proven effective both in caspase-8-deficient and FADD-overexpressing HNSCC cells, inducing programmed necrosis and increasing cell radiosensitivity after X-ray treatment (Refs Reference Uzunparmak129, Reference Eytan130).

Although ATG proteins are often upregulated in tumour cells, the activation of this specific pathway could lead to massive cell death after radiotherapy. The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway is a critical regulator of autophagy, and targeting this could be an important therapeutic strategy enhancing the sensitivity of tumour cells to IR. Several pharmacological inhibitors have been tested on various cancer cell types, including HNSCC cells, both in vitro and in xenograft models with promising results (reviewed in (Ref. Reference Wanigasooriya131)). A phase I clinical trial is currently ongoing in high-risk patients with locally advanced squamous cell carcinoma to test a potent and highly specific oral pan-class I PI3K inhibitor, BKM120 (ClinicalTrials.gov Identifier NCT02113878). Other ATG targets have been investigated, including Kelch-like ECH-associated protein 1 (Ref. Reference Deville80) and p62, whose overexpression has been reported to induce autophagy in HNSCC cells (Ref. Reference Lee132).

The induction of senescence can be clinically beneficial, but currently there are no specific senescence-inducing agents available. The ability of cells to enter radiation-induced senescence is almost strictly linked to p53. In fact, it has been reported that HNSCC cells with wild-type or non-disruptive mutations of p53 undergo senescence after radiotherapy, while p53 mutant cells are more radioresistant and show higher levels of senescence markers after treatment with metformin (anti-diabetic agent that has been shown to induce reactive oxygen species) (Ref. Reference Skinner103). Reactivation of p53 restored senescence and increased radiosensitivity in p53 mutant HNSCC cells, although interestingly the study reported that the mechanism is only partially p53-dependent (Ref. Reference Chuang133). Treatment with rapamycin or the mTOR inhibitor PP242 in parotid carcinoma cells in vitro increased heterochromatin formation, and induced irreversible growth arrest and premature senescence. Whilst in tumour xenografts, PP242 delayed tumour regrowth after X-ray irradiation and increased senescence-associated β-galactosidase staining (Ref. Reference Nam134).

Inhibition of proteins involved in DNA replication and mitosis could be a valid therapeutic solution to force the cells into mis-segregation of chromosomes and promote aberrant mitotic division. As mitotic catastrophe is a relatively new cellular endpoint, the current literature is scarce and only a few candidates have been investigated as chemotherapeutic targets. Polo-like kinase 1 (PLK1) is a serine/threonine kinase which functions as a pleiotropic master regulator of mitosis and regulates DNA replication after stress (Ref. Reference Joukov and De Nicolo135). PLK1 depletion has been reported to induce mitotic cell cycle arrest and inhibit the separation of sister chromatids in oesophageal cancer cells, causing failure of cytokinesis followed by massive apoptotic cell death (Ref. Reference Bu136). These results have been confirmed in human nasopharyngeal cancer cells, where PDK1 inhibition was found to greatly reduce cell survival, alone or in combination with radiation, due to G2/M cell cycle arrest and aberrant spindle formation, which in turn caused mitotic catastrophe (Ref. Reference Shi137). Furthermore, co-treatment with PLK1 inhibitor and inhibitors targeting Aurora A and Aurora B enhanced metaphase arrest and mitotic slippage in nasopharyngeal cancer cells, ultimately inducing mitotic catastrophe (Ref. Reference Li138). The Aurora A and B protein kinases are key players in mitotic control and therefore another set of potential targets to increase cancer radiosensitivity. Aurora B inhibition in fact has been demonstrated to lead to G2/M accumulation, polyploidy and subsequent cell death by mitotic catastrophe in anaplastic thyroid carcinoma in vitro and reduced growth of ATC tumour xenogratfs (Ref. Reference Libertini139). Aurora A depletion appears to have a limited effect on HPV-negative HNSCC cells when administered alone, although still causing spindle defects and cytostasis, while co-treatment with a WEE1 cell cycle checkpoint kinase inhibitor triggered mitotic catastrophe in vitro and reduced tumour growth in FaDu and Detroit 562-derived xenografts (Ref. Reference Lee140). Other potential cellular targets for inhibitors that have yielded promising results as chemotherapeutic agents against HNSCC cells include WEE1 (Refs Reference Mak141–Reference Deneka143), CHK1/2 (Refs Reference Mak141, Reference Deneka143) and PP2A (Ref. Reference Lv86), although further studies are needed in order to determine their specific potential as radiosensitisers.