Review

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Head and neck squamous cell carcinoma

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EGFR-PI3K-AKT-mTOR

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signaling in HNSCC 3.

Radioresistance in HNSCC

4.

EGFR-PI3K-AKT-mTOR pathway as a target to overcome radioresistance in HNSCC

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Expert opinion

Targeting EGFR-PI3K-AKT-mTOR signaling enhances radiosensitivity in head and neck squamous cell carcinoma Dominik Horn, Jochen Hess, Kolja Freier, Ju¨rgen Hoffmann & Christian Freudlsperger† †

University Hospital Heidelberg, Department of Oral and Maxillofacial Surgery, Heidelberg, Germany

Introduction: Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by high resistance to radiotherapy, which critically depends on both altered signaling pathways within tumor cells and their dynamic interaction with the tumor microenvironment. Areas covered: This review covers EGFR-phosphoinositide 3-kinase (PI3K)protein kinase B (AKT)-mechanistic target of rapamycin (mTOR) signaling in HNSCC. The role of each pathway node in radioresistance is discussed. Preclinical and clinical innovative aspects of targeting EGFR-PI3K-AKT and mTOR are demonstrated. Ongoing clinical trials and future perspectives are presented. Expert opinion: Different cellular signaling pathways seem to mediate radioresistance in advanced HNSCC and various molecular targeted therapies are currently being investigated to sensitize tumor cells to radiotherapy. Recently, new insights in the mutational landscape of HNSCC unraveled critical alterations in putative oncogenes and tumor suppressor genes and have emphasized the importance of PI3K and the corresponding upstream and downstream signaling pathways in pathogenesis and treatment response. The frequent activation of the EGFR-PI3K-AKT-mTOR pathway in HNSCC and its implication in the context of radiosensitivity make this pathway one of the most promising targets in the therapy of HNSCC patients. Clinical studies targeting EGFR and mTOR in combination with radiotherapy are under investigation. Keywords: head and neck squamous cell carcinoma, mechanistic target of rapamycin, proteinkinase B, phosphoinositide 3-kinase, radiotherapy Expert Opin. Ther. Targets [Early Online]

1.

Head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC), an entity that comprises epithelial cancer of the oral cavity, oropharynx, hypopharynx and larynx is the sixth leading cancer by incidence worldwide [1]. Beyond tobacco and alcohol abuse, the oral infection by high-risk types of human papillomavirus (HPV) is confirmed as an additional risk factor in cancer etiology [2-5]. This evidence resulted in new subgroups of HNSCC (HPV-positive vs HPV-negative) with distinct molecular and cellular characteristics, epidemiology and prognosis [6-9]. As early stages of HNSCC are generally curable with primary surgery, prognosis for patients with locally advanced or metastatic disease is still dismal with a 5-year survival rate of < 50% [10]. Whereas advances in surgery and chemoradiation have provided better organ preservation, only modest improvement in the 5-year survival of HNSCC patients has been achieved in the last decades [11,12]. Locoregional recurrence remains the major form of treatment failure as up to 60% of patients develop local recurrence 10.1517/14728222.2015.1012157 © 2015 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted

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D. Horn et al.

Article highlights. . . . . .

Activation EGFR-PI3K-AKT-mTOR pathway is a frequent event in HNSCC. Radioresistance is a major challenge in HNSCC therapy. EGFR-PI3K-AKT-mTOR signaling plays a key role in radioresistance. Experimental inhibition of pathway components leads to radiosensitivity. PI3K, AKT, mTOR or dual inhibitors are promising therapeutics in individualized HNSCC therapy.

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This box summarizes key points contained in the article.

commonly resistant to radiotherapy [13]. Tumor-cell proliferation, intrinsic mechanisms and micro-environmental interactions are implicated in radioresistance. But so far, little is known about the underlying molecular processes and resistance to radiotherapy remains a major challenge in the treatment of advanced HNSCC. Moreover, the interdisciplinary therapy is confronted with an increasing subgroup of younger patients with HPV-related tumors induced by molecular alterations that differ from HPV-negative HNSCCs. Therefore, novel strategies are required to understand the characteristics of resistant cells in predicting response to therapy and in reducing toxicity by the use of targeted drugs that sensitize tumor cells to radiation. Genomic characterization of HNSCC has demonstrated four common driver-signaling pathways: mitogenic signaling, NOTCH/differentiation, cell cycle, and p53/apoptosis, leading to different approaches of potential therapeutic targets [14-17]. In addition, several intracellular signaling pathways have been implicated in mediating resistance or sensitization to radiotherapy, where the EGFR-phosphoinositide 3-kinase (PI3K)-proteinkinase B (AKT)-mechanistic target of rapamycin (mTOR) pathway seems to play a key role [18-20]. The EGFR-PI3KAKT-mTOR pathway is by far the most altered mitogenic pathway, as > 80% of HNSCC tumors in general harboring molecular alterations in one or more components of this pathway [15,20]. Especially HPV-infected oropharyngeal tumors show frequent alterations in the PI3K pathway with activation of the downstream target mTOR [5,21]. This review focuses on the central nodes within this pathway including EGFR, PI3K, phosphate and tensin homolog deleted on chromosome 10 (PTEN), 3-phosphoinositidedependent protein kinase 1 (PDK1), AKT and mTOR as well as their potential role in the context of radiosensitivity in HNSCC. 2.

EGFR-PI3K-AKT-mTOR signaling in HNSCC

The EGFR-PI3K-AKT-mTOR signaling pathway plays an important role in numerous cellular processes, including proliferation, growth, differentiation, migration, inflammation and survival under normal physiological and pathophysiological conditions, including cancer [22]. 2

The EGFR is the cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands [23]. Multiple ligands activate EGFR, which leads to the activation of class I PI3K. Once activated, PI3K phosphorylates phosphotatidylinositol-4,5-bisphosphate to generate phosphatidylinositol-3,4,5-trisphosphate (PIP3) (Figure 1) [22]. PTEN, an important tumor suppressor, antagonizes the PI3K function [24]. PIP3 initiates AKT activation by its translocation to the plasma membrane, leading to a conformational change. Subsequently, AKT is phosphorylated at three regulatory sites: At Thr308 by PDK1 [25], at Ser473 by the rapamycin insensitive companion of mTOR complex [26] and at Ser129 by casein kinase 2 [27]. Once activated, AKT phosphorylates multiple downstream targets, including the antiapoptotic NF-kB pathway [28], FOXO family members [29], and mTOR complex 1 (mTORC1) [30]. High activation of EGFR-PI3K-AKT-mTOR signaling in HNSCC, due to various genetic and epigenetic mechanisms, including gain-of-function mutations in PIK3CA (6 -- 13%), PIK3CA amplification (20%), PIK3CA gene overexpression (52%), LOH of PTEN (85%), reduced PTEN protein expression (30%), and inactivating mutations of PTEN (4%) has been confirmed by several authors [15-17,21]. Consequentially, high activation status of EGFR-PI3K-AKT-mTOR signaling was linked to worse clinical outcome in patients with HNSCC [31]. Key nodes of EGFR-PI3K-AKT-mTOR signaling, which play an important role in mediating radioresistance in HNSCC and therefore present attractive targets for radiosensitzing HNSCC tumors, are described in the following sections. Clinical studies currently investigating the radiosensitzing effect of EGFR-PI3K-AKT-mTOR pathway inhibition in HNSCC are summarized in Table 1. 3.

Radioresistance in HNSCC

Besides surgery, radiotherapy is the most important treatment modality in HNSCC. Irradiation induces free radicals as a result of ionizations in the DNA, causing DNA doublestranded breaks or the induction of reactive oxygen species in target tissues. Oxygen molecules react with free radicals and stabilize the DNA damage, which is the molecular basis for response to radiotherapy [32]. Malignant cells can achieve radioresistance by several escape strategies. One reason for therapy failure is enhanced tumor cell proliferation [33]. Accelerated repopulation can occur during radiotherapy by surviving cells inducing tumor recurrence [34]. Due to accelerating and fractionating radiation dose as well as the combination with chemotherapy or hypoxic modulation, radiosensitization has been achieved [35-37]. An in vitro study of a HNSCC cell line showed concurrent increase of EGFR expression with accelerated tumor cell repopulation 3 weeks after irradiation [38]. Furthermore, tumor cells are capable of DNA damage repair by DNA-protein kinases (DNAPK). Activation of EGFR downstream pathways has been found to be responsible for increased resistance to DNA damaging agents [39].

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EGFR-PI3K-AKT-mTOR signaling and radiosensitivity in HNSCC

EGFR PI3K

PIP3

PIP2

Thr308

P

PDK1

P

CK2

AKT

mTORC2 P

mTOR

Ser473 Ser129

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Rictor

mTORC1 Autophagy

mTOR

NF-κB

Raptor

Inflammatory response

Survival

FOXO

Apoptosis

Cell proliferation

Figure 1. Current understanding of EGFR-PI3K-AKT-mTOR signaling: ligand binding to the EGFR activates class I PI3K leading to phosphorylation of phosphotatidylinositol-4,5-bisphosphate (PIP2) to generate phosphatidylinositol-3,4,5-trisphosphate (PIP3). Phosphate and tensin homolog deleted on chromosome 10 (PTEN) antagonizes the PI3K function. PIP3 initiates AKT activation followed by phosphorylation 3 regulatory sites: At Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1), at Ser473 by the mTOR-RICTOR (rapamycin insensitive companion of mTOR) complex and at Ser129 by casein kinase 2 (CK2). Once activated, AKT phosphorylates multiple downstream targets, including the anti-apoptotic NF-kB pathway, FOXO family members, and mTOR complex 1 (mTORC1). AKT: Proteinkinase B; mTOR: Mechanistic target of rapamycin; PI3K: Phosphoinositide 3-kinase.

There is ongoing evidence that the microenvironement influences response to ionizing radiation [34,40]. For example, hypoxic conditions in tumors are associated with treatment failure and worse patient outcome [41,42]. Malignant cells can adapt to hypoxic conditions by activating survival and proliferation pathways, which induce among others the transcription factor hypoxia-inducible-factor (HIF)-1 [43,44]. HIF-1 can increase gene expression of survival, angiogenesis and migration mechanisms [45,46]. The complex interaction of the tumor microenvironement is mostly unidentified and is under current investigation. In the emergence of radioresistance most molecular processes share the involvement of EGFR-PI3K-AKT-mTOR signaling. Therefore molecular therapies targeting EGFRPI3K-AKT-mTOR pathway are of high importance.

EGFR-PI3K-AKT-mTOR pathway as a target to overcome radioresistance in HNSCC

4.

EGFR The EGFR is a well-characterized proto-oncogene that is activated in multiple cancers, where it has been shown to promote 4.1

tumor progression. In addition, EGFR is overexpressed in the majority of HNSCC patients [47] and has been linked to poor prognosis and survival rates [48,49]. One reason for the overexpression is a chromosomal amplification, as cytogenetic analysis of HNSCC tumors showed increased copy numbers of 7p12 (the locus for EGFR) in 30 -- 47% of samples [50,51]. The presence of EGFR amplification was associated with poor survival [52]. However, the main mechanism of constitutive EGFR up-regulation seems to be transcriptional activation [48]. One of the most common EGFR alterations in several cancers, including HNSCC, consists of a truncation in the extracellular domain known as EGFR variant III (EGFRvIII). This mutation eliminates exons 2--7 and results in a distorted ligand-binding region [53,54]. EGFRvIII does not bind ligands but is constitutively activated in a ligand-independent manner. In gliomas, where it has been most extensively studied, EGFRvIII expression correlates with increased tumorigenicity in mouse models [55] and poor prognosis in the clinical setting [56]. Furthermore, EGFRvIII has been reported to increase resistance to anti-tumor agents, including EGFR inhibitors [57]. Moreover, the expression of EGFRvIII is unique to cancer, as EGFRvIII has not been observed in normal tissue. In HNSCC, this constitutively active mutation is reportedly

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Table 1. Overview of clinical studies investigating the radiosensitzing effect of EGFR-PI3K-AKT-mTOR pathway inhibition in HNSCC. Target

Drug name

EGFR*

Cetuximab Nimutuzumab

PI3K AKT mTOR

Zalutumumab Gefitinib Erlotinib Everolimus

Proteasome

Bortezomib

Combination RT RT RT RT RT RT RT RT RT RT RT RT

+ + + + +

Platin-based Platin-based Platin-based Platin-based Platin-based

Phase

CH CH CH CH + cetuximab CH + cetuximab

III II IIb III II II I I I I I I

Status Terminated, results Terminated, results Terminated, results Active, not recruiting Withdrawn Terminated Terminated Completed Terminated Completed Completed Completed

Ref. [122] [67] [68]

NCT00496652 NCT00233636 NCT01192815 NCT01058408 NCT00858663 NCT01057277 NCT01445405 NCT00629226 NCT00011778

*Listed EGFR targeting studies are reduced to radiation as single adjuvant due to the variety of studies. AKT: Proteinkinase B; CH: Chemotherapy; EGFR: Epidermal growth factor receptor; HNSCC: Head and neck squamous cell carcinoma; mTOR: Mechanistic target of rapamycin; PI3K: Phosphatidyl-inositol-3-kinase; RT: Radiotherapy.

found in > 42% of HNSCC tumors and enhances tumor growth as well as resistance to EGFR-blocking antibodies in vitro and mouse xenograft models [57,58]. Therapeutic approaches for interrupting EGFR signaling include monoclonal antibodies targeting the ligand-binding domain of the receptor and small-molecule tyrosine kinase inhibitors [59]. The most studied monoclonal antibody is cetuximab (Erbitux, ImClone Systems, New York, NY, USA). It received an FDA approval in 2006 for the treatment of locally or regionally advanced HNSCC in combination with radiotherapy [60] and as a single agent for the treatment of platinum-refractory recurrent/metastatic HNSCC [61]. However, cetuximab as a single agent in patients with HNSCC typically shows response rates of only 13% [61], along with toxicities of diarrhea, skin rash and infusion reactions. In one study arm of a randomized Phase IIb study in India with unresectable HNSCC, patients received radiotherapy with or without the monoclonal EGFR antibody nimotuzumab (h-R3, BIOMAb EGFR, Biocon, Bangalore, India). In contrast to Cetuximab, h-R3 showed less toxic side effects, especially no severe skin rashes [62]. As the most frequent side effect, skin rashes occur in up to 90% of the patients treated with cetuximab, whereas 15% develop severe skin reactions [63-65]. As the reason for its reduced toxicity, the lower receptor binding affinity of nimotuzumab is proposed [66]. Patients treated with nimotuzumab showed a better but not significant progression-free survival and overall survival [67]. Another randomized Phase II study with 106 patients, investigating the additional effect of nimotuzumab to radiotherapy, could show a trend towards a survival benefit, especially in EGFR-positive tumors in contrast to EGFR-negative tumors [68]. This study highlights the importance of 4

stratifying patients according to the molecular landscape of the respective tumor. In the past, it has been discovered that EGFR overexpression might negatively affect tumor response to radiotherapy. Sheridan et al. discovered that HNSCC cells with high levels of EGFR expression showed a higher radioresistance compared to cells with low EGFR expression [69]. In addition, Milas et al. demonstrated that overexpression of EGFR is linked to increased radioresistance [70], which was confirmed for HNSCC [71]. Ang et al. showed in a prospective multicenter clinical trial of the Radiation Therapy Oncology Group that EGFR expression was highly significantly correlated with poorer overall and lower disease-free survival rates in patients treated with a consistent radiation regimen [72]. Prolonged exposure of HNSCC cells to EGF increased radiosensitivity, which was mediated through EGF-induced EGFR degradation [73,74]. In addition, Balaban et al. showed that monoclonal antibodies targeting EGFR increased radiationinduced apoptosis [75]. Furthermore, radiotherapy leads to increased EGFR signaling and subsequent stimulation of downstream signaling pathways, including PI3K-AKT signaling [76-79]. In addition, radiation leads to increased levels of phosphorylated EGFR [76-79] and induces EGFR translocation into the nucleus, where it increases the DNAPK activity, leading to enhanced DNA-damage repair capability. Cetuximab was shown to inhibit EGFR nuclear translocation and activation of DNAPK (Figure 2) [80]. These findings provide compelling clinical evidence in support of experimental data showing that EGFR expression is a strong and consistent indicator of cellular radioresistance in vitro [69] and strengthen the close relationship between EGFR overexpression and tumor radioresistance in vivo [75,81]. Moreover, EGFRvIII expression decreased HNSCC cell apoptosis in response to cisplatin and

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EGFR-PI3K-AKT-mTOR signaling and radiosensitivity in HNSCC

EGFR inhibitor cetuximab wtEGFR

EGFRvIII

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EGFR phosphorylation P

P

PI3K-AKT

Nuclear translocation

Hif-1

P EGFR

Cell survival

DNAPK

Figure 2. The role of wtEGFR and EGFRvIII in the context of radiosensitivity: radiation leads to increased levels of phosphorylated wtEGFR and induces EGFR translocation into the nucleus, where it increases the DNA-protein kinase (DNAPK) activity, leading to enhanced DNA-damage repair capability, which can be blocked by the monoclonal antibody cetuximab. EGFRvIII-expressing cancer cells show hyperactivated PI3K-AKT signaling, leading to elevated DNA-damage repair capability through activation of DNAPK. AKT: Proteinkinase B; EGFRvIII: EGFR variant III; PI3K: Phosphoinositide 3-kinase.

induced resistance to the growth inhibitory effects of cetuximab in vitro and in vivo [57]. Some reports have indicated that activated pathways downstream of EGFRvIII are responsible for treatment failure. It has recently been shown that cancer cells expressing EGFRvIII might be partly radioresistant due to increased PI3K-AKT signaling (Figure 2) [82]. Most notably, EGFRvIII-expressing cancer cells show elevated DNA-damage repair capability through activation of DNAPK [83] and EGFRvIII displays even higher levels of radiation-induced activation of DNAPKs compared with the wild-type receptor [84]. This is possibly transduced via hyperactivated PI3K-AKT signaling, as EGFRvIII preferentially activates PI3K-AKT, whereas ligand-activated wild-type EGFR stimulates both the Ras-Raf-MAPK and PI3K-AKT pathways [85].

PI3K-AKT and PTEN The PI3K pathway has been shown to be one of the most frequently mutated pathways in HNSCC. Recent studies on the mutational landscape of HNSCC showed alterations at the PI3K pathway in up to 30.5% of all tumors. Interestingly, mutations in PI3K genes are more common in HPV-positive HNSCC [20]. In oral HNSCC, high expression of the 4.2

downstream target AKT is correlated to worse prognosis and poor local control in different studies [31,86]. Several mechanisms, such as intrinsic cellular resistance and microenvironmental interactions, seem to be involved in PI3K-AKT-mediated resistance to irradiation [87]. Activation of the PI3K-AKT pathway through EGFR or RAS upstream signaling increases the survival of tumor cells in vitro, implicating PI3K-AKT as an important mainstay in radioresistance [39]. DNAPKs are responsible for repair mechanisms and hence cell survival, in which the PI3K-AKT pathway is involved through upstream EGFR signaling [88,89]. A known mechanism for arising radiation resistance is hypoxia. In general, low oxygen levels decelerate cell metabolism, resulting in less proliferation [90]. Hypoxia-resistant cells as a clonogenic subpopulation might survive and repopulate after treatment [91]. Low oxygen levels in HNSCC promote more invasive phenotypes [92] and correlate with poor local control after radiation [93,94]. Blocking EGFR and hence PI3K-AKT signaling, led to improved oxygenation [95,96]. Constitutively active AKT favors hypoxia-inducible transcription factor 1 (HIF-1)-dependent gene transcription, which modulates angiogenesis, pH, and glucose metabolism (Figure 2) [97,98]. Deletion of PTEN leads to permanent activation of the PI3K pathway, which has been shown to contribute to

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D. Horn et al.

chemo- and radioresistance in cancer stem cells, emphasizing the importance of microenvironemental interactions [99,100]. Furthermore, malignant epithelial cells in HNSCC can undergo epithelial to mesenchymal transition (EMT), resulting in a phenotype of cells with overexpression of MMPs and down-regulated E-cadherin, which is associated with low response to irradiation. EMT induction is mediated through histone deacetylase via upregulation of AKT [101,102]. PI3K can be targeted by small molecules (e.g., preclinical use: LY29004, wortmannin and K.SF1126; under clinical investigation: BKM120, PX-866 and BYL719). Irreversible inhibition of PI3K by LY294002 leads to radiosensitization in astrocytic cells [103,104] as well as in cell lines of HNSCC [31]. We could find similar effects in an ex vivo tumor culture model that preserves the microenvironment in primary and recurrent HSNCC [105]. This preclinical tool for evaluation of therapeutic drugs offers an additional option to understand the complex microenvironmental interactions leading to radioresistance. Direct targeting of AKT is an alternative for therapeutic inhibition. For example, perifosine and triciribine are experimental small molecule drugs to inhibit the phosphorylation of AKT. We could show in ex vivo cultures that inhibition of AKT by triciribine led to an improved radiosensitivity similar to LY294002, but showed no effect on cell proliferation when used as a single agent [105]. Up to date there is only one clinical study that applies an AKT inhibitor (MK2206) in Phase II in HNSCC (NCT01349933). Targeting PTEN as a negative regulator of PI3K-AKT signaling is an additional working point for cancer therapy. Bortezomib is a proteasome inhibitor that targets the 26S proteasome. Bortezomib blocks the proteasomemediated degradation of NF-kB [106], and PTEN with a net effect of decreased PI3K-AKT signaling, and potentially enhancing radiosensitivity [107-109]. A Phase I clinical study in 27 patients with recurrent disease showed tolerable toxic side effects, when combining bortezomib with chemoradiotherapy [110]. Table 1 shows ongoing trials combining bortezomib with radiotherapy. There are few clinical studies published using PI3K or AKT inhibitors in HNSCC and none of them combines those small molecule inhibitors with irradiation (Table 1). Only few Phase I and II studies for HNSCC are confined on PI3K inhibitors with conventional chemotherapeutics or cetuximab [111,112]. Regarding PTEN, ongoing clinical studies using bortezomib in combination with irradiation will bring new information about this promising target therapy, in particular the role of PTEN expression as a potential biomarker for bortezomib response. For the investigation of the sensitizing effect of selective PI3K and AKT inhibitors on radiotherapy, further clinical studies are urgently needed. mTOR Ekshyyan et al. demonstrated that the mTOR1 inhibitor temsirolimus radiosensitise HNSCC cells in vitro and in vivo [113]. In their study, combination of temsirolimus and radiation was significantly more effective in reducing tumor growth compared to combination of radiation and cisplatin. This effect 4.3

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was mediated through reduction of radiation-induced activation of the mTOR pathway by temsirolimus, which led to increased apoptosis and decreased number of tumor blood vessels. Ongoing clinical trials investigating the combination of everolimus or temsirolimus with radiation in HNSCC are summarized in Table 1. In addition, Cerniglia et al. investigated the effect of the selective dual pan-class I PI3K and mTOR kinase inhibitor NVP-BEZ235 in HNSCC [114]. NVP-BEZ235 significantly increased radiosensitivity of HNSCC cells in vitro and using xenografts. In particular, NVP-BEZ235 decreased the process of DNA-damage repair by attenuating radiation-induced phosphorylation of DNAPKs. Interestingly, NVP-BEZ235 induced autophagy in cells, and adding autophagy inhibitors to the combination of NVP-BEZ235 and radiation increased cell death, indicating a promising combination strategy for further investigations [114]. Autophagy, the phenomenon of cellular selfdigestion has the potential to promote either cell survival or cell death in the context of different cancers and postirradiation [115] and seems to be mediated through the mTORC1 (Figure 1) [116]. Furthermore, in radiation-resistant cancer cells, blocking PI3K-AKT-mTOR signaling using NVP-BEZ-235 increased radiosensitivity through augmented autophagy [117]. 5.

Expert opinion

Acquired resistance to radiotherapy is a major drawback in the treatment of HNSCC, especially with regard to the high rate of locoregional recurrence of up to 60% [13]. Hence, understanding the molecular mechanisms associated with radioresistance in cancer cells will lead to improvements in patient survival. Multiple studies have revealed that components of the EGFR-PI3K-AKT-mTOR pathway are mutated in a considerable percentage of HNSCC tumors. Especially, the recent genomic studies investigating the mutational landscape of HNSCC showed high rates of PI3K alterations. Cell signaling via the EGFR-PI3K-AKTmTOR pathway plays a crucial role in the mainstay of radioresistance in HNSCC, although the exact molecular mechanisms are still unknown. EGFR is currently the most attractive molecular target for radiosensitizing HNSCC, as EGFR is overexpressed in the majority of HNSCC. Anti-EGFR agents augment effects of radiation by abrogating EGFR activation and translocating EGFR into the nucleus, thus preventing DNA damage repair. The approval of cetuximab by the FDA and EMA, for the treatment of locally advanced disease in combination with irradiation, introduced a new era of molecular targeting in HNSCC. Downstream of EGFR, the PI3K-AKT pathway is an attractive target for molecular therapy due to its role in radioresistance. However, there are currently no clinical studies evaluating PI3K or AKT inhibitors as single adjuvant to radiotherapy in HNSCC. The recent genomic studies

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EGFR-PI3K-AKT-mTOR signaling and radiosensitivity in HNSCC

identifying PI3K alterations as a frequent event in HNSCC fuelled the interest of targeting these elements in the cellular network to hopefully overcome radioresistance or at least radiosensitize HNSCC. Sensitization, and therefore reduction of toxicity, is the goal of the ongoing RTOG 1016 trial comparing cisplatin versus cetuximab with radiotherapy in HPVpositive tumors. Lechner et al. have separated HPV-positive and HPV-negative HNSCC tumors on the molecular level. In HPV-positive tumors, genes were identified implicated in frequent activation of the PI3K-AKT-mTOR pathway. PIK3CA mutation and PTEN inactivation by gene copy loss or mutation were detected in > 60% of HPV-positive HNSCC [118]. Experimental in vitro and in vivo studies showed antitumor efficiency of mTOR inhibitors and could prolong survival in a xenograft model [119,120]. Current ongoing clinical trials in terms of mTOR inhibition will deliver more information about these promising antitumor effects (Table 1). As PI3K mutation and PTEN activation were detected in 31% of HPV-negative HNSCC, adjuvant targeting PI3K-AKT-mTOR in combination with radiotherapy might be an option to reduce toxicity of treatment in older and multimorbid patients in this subgroup. In terms of the increasing number of younger patients, inhibiting these kinases could reduce the cumulative radiation dose expecting a better organ preservation and a lower risk of radiation-related secondary diseases. An issue for the clinical use of EGFRPI3K-AKT-mTOR targeted therapy is an individual approach due to varying molecular alterations and the medical condition of each patient. Specific biomarkers could identify patients considering the response rate to inhibitors. Recently, Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers.

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p62/SQSTM1 was identified as a potential biomarker for the sensitivity to PI3K inhibitors [121]. In conclusion, there are promising preclinical results for radiosensitation HNSCC by targeting the PI3K/AKT/ mTOR pathway. We could enlarge the tools for the evaluation of pathway inhibitors in combination with radiotherapy with an ex vivo culture system respecting the microenvironment. Biomakers could help to determine individual response to PI3K/AKT/mTOR therapy. Various mono or dual inhibitors are under clinical investigation for HNSCC. Besides EGFR inhibition, everolimus as an mTOR inhibitor is the most promising candidate for a successful therapeutic combination with radiotherapy. The partly ongoing Phase I studies (NCT01058408, NCT00858663, NCT01057277) and the preclinical results will hopefully be transferable into Phase II and III clinical studies. PI3K and AKT inhibition is a potential intervention to radiosensitize HNSCC in clinical studies. Concerning the mono or dual PI3K/AKT inhibitors the preclinical data is strong enough to evaluate these inhibitors in Phase I with the objective to sensitize HNSCC to irradiation and to finally overcome radioresistance.

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Affiliation Dominik Horn1, Jochen Hess2,3, Kolja Freier1, Ju¨rgen Hoffmann1 & Christian Freudlsperger†1 † Author for correspondence 1 University Hospital Heidelberg, Department of Oral and Maxillofacial Surgery, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany Tel: +49 0 6221 56 38462; Fax: +49 0 6221 56 4222; E-mail: [email protected] 2 University Hospital Heidelberg, Section Experimental and Translational Head and Neck Oncology, Department of Otolaryngology, Heidelberg, Germany 3 German Cancer Research Center (DKFZ), Research Group Molecular Mechanisms of Head and Neck Tumors, Heidelberg, Germany

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Targeting EGFR-PI3K-AKT-mTOR signaling enhances radiosensitivity in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by high resistance to radiotherapy, which critically depends on both altered...
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