Schuurbiers, Olga C.J. MD*; Kaanders, Johannes H.A.M. MD, PhD†; van der Heijden, Henricus F.M. MD, PhD*; Dekhuijzen, Richard P.N. MD, PhD*; Oyen, Wim J.G. MD, PhD‡; Bussink, Johan MD, PhD†
Lung cancer is one of the most lethal forms of cancer, frequently presenting in advanced stages. Despite combined treatment modalities prognosis is poor with 5-year survival rates of 10 to 15%. In combined modality treatment the phosphatidylinositol-3-kinase (PI3-K)/protein kinase B (AKT) pathway (Figure 1) is involved in resistance mechanisms for radiotherapy and for chemotherapy. The PI3-K/AKT cascade can be up-regulated by radiotherapy and plays a central role in the three major radiotherapy resistance mechanisms in non-small cell lung cancer (NSCLC) and other tumors: intrinsic radiosensitivity, tumor proliferation, and tumor cell hypoxia.1 Understanding this pathway offers opportunities to unravel mechanisms of radioresistance. Furthermore, specifically targeting this pathway can potentially enhance the efficacy of combined treatment modalities. In this review we discuss the potential effects of inhibition of the PI3-K/AKT pathway in the light of the tumor microenvironment, more specifically, what the effect may be on tumor cell hypoxia, proliferation, and DNA-repair in NSCLC.
STRATEGIES FOR INCREASING RESPONSIVENESS TO RADIOTHERAPY
Modifications in fractionation schedules have been developed to overcome radiation resistance. Hyperfractionation is designed to overcome intrinsic radioresistance and exploits the difference in the fractionation sensitivity between rapidly and slowly renewing tissues, i.e., a higher total dose can be given when the dose per fraction is reduced. However, in unresectable locally advanced NSCLC patients (95% stage III disease) hyperfractionated radiotherapy failed to demonstrate improvement over conventional radiotherapy.2 Clinical studies have shown the relevance of tumor cell repopulation on tumor control and survival after radiotherapy in NSCLC. Delays exceeding 5 days beyond planned radiation treatment duration led to pronounced reduction in 2- (33 and 15%) and 5-year survival rates (14 and 0%) for locally advanced NSCLC.3,4 Accelerated fractionation aims to counteract compensatory tumor cell repopulation by reducing the overall treatment time. A multicenter randomized controlled trial of continuous hyperfractionated accelerated radiotherapy versus conventional radiotherapy was conducted in 563 inoperable NSCLC patients (61% stage III disease). With this treatment strategy an improvement of 2-year survival from 20 to 29% was achieved.5 This illustrates the importance of proliferation because this improvement was obtained despite a reduction of the total dose by 10%.
Another approach to increase radiosensitivity is to combine radiotherapy with chemotherapy. Randomized trials report modest improvements in survival for concurrent chemoradiotherapy: 5-year survival of 17% for concurrent versus 6% for sequential chemoradiotherapy in the Cancer and Leukemia Group B trial.6 Data from three trials were used in a meta-analysis comparing concurrent versus sequential chemoradiotherapy. The results showed a significant reduction (14%) in the 2 year risk of death if chemotherapy was given together with radiotherapy (RR 0.86; 95% CI 0.78–0.95; p = 0.003).7
The third radioresistance mechanism, hypoxia, can be overcome by combining radiotherapy with treatments such as hyperoxic gas breathing, nitroimidazole radiosensitizers, or hypoxic cytotoxins.8 Although these approaches were successful in head and neck cancer and cervical cancer, data from patients with lung cancer are limited.8 In advanced NSCLC a randomized trial of tirapazamine, a hypoxic cytotoxin, with cisplatin versus cisplatin alone showed significantly improved overall response rate (28 versus 14%) and 1-year survival rate (34 versus 23%) for the combination.9
PI3-K is a cytosolic complex and consists of an 85 kDa regulatory subunit and a 110 kDa catalytic subunit (p110α). Upon ligand-mediated activation of receptor TK the p85–p110 complex is recruited to the receptor where it is activated and generates phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP3 functions as a second messenger and recruits AKT to the cell membrane where it binds by its pleckstrin-homology-domain.10–12 At the membrane, AKT is phosphorylated (pAKT) at threonine 308 (Thr308) by 3-phosphoinositide-dependent protein kinase-1 and thereby activated.13 Full activation requires additional phosphorylation at serine 473 (Ser473) by PDK2. After activation, AKT translocates to the cytosol and nucleus to phosphorylate its substrates.14
PI3-K/AKT is one of the major downstream targets of the ErbB tyrosine kinase receptor family (Figure 1). Activation of this pathway is not only achieved after activation of the epidermal growth factor receptor (EGFR) but also, and more effectively through other members of the ErbB-membrane receptor family. The four members of this family, ERBB1 (EGFR), ERBB2 (HER-2/neu), ERBB3 (HER-3), and ERBB4 (HER-4) are activated upon ligand-induced (e.g., epidermal growth factor or tumor growth factor α) receptor homo- and heterodimerization.15 Upon heterodimerization, the cytoplasmatic domain of ERBB-3 undergoes tyrosine phosphorylation, and activates PI3-K. EGFR itself is a weak direct activator of PI3-K but in particular connects to the RAS/PI3-K/AKT pathway or collaborates with ErbB-3. It has been demonstrated that PI3-K/AKT signaling is tightly regulated by EGFR in some tumors16 but this is not a general feature and EGFR overexpression is not necessarily associated with PI3-K/AKT activation.15,17
Furthermore, receptor TK independent activation of the PI3-K/AKT pathway is commonly observed in many cancers as well, and can occur through multiple mechanisms, such as mutation or amplification of PI3-K, amplification of AKT, activation of oncogenes upstream (e.g., RAS), or mutation or decreased expression of the tumor suppressor protein Phosphatase and TENsin homolog (PTEN).18 The PTEN tumor suppressor gene is the central negative regulator of the PI3-K/AKT pathway by dephosphorylation of PIP3 at the plasma membrane. It was demonstrated that loss of PTEN in EGFR expressing tumor cells counteracts the antitumor action of EGFR inhibitors by permitting AKT activity independent of receptor TK inputs.19 PTEN is also located in the nucleus and maintains chromosomal stability through association with centromere protein in a phosphatase independent manner and through control of DNA double strand break repair mechanisms.20,21
The PI3-K/AKT pathway is frequently over-activated in many solid tumors such as NSCLC.11,12,17 PI3-K/AKT activation triggers a cascade of responses, which has consequences for all major cancer-cell growth mechanisms: survival, proliferation, and cell growth. Additional effects involve DNA double strand break repair and tumor angiogenesis through hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF).12
THE PI3-K/AKT-PATHWAY AND RADIATION RESISTANCE
The PI3-K/AKT Pathway and Intrinsic Radiosensitivity
Radiation induced activation of multiple signaling pathways, depends at least in part on the expression of the EGF-receptor. In general, this radiation induced signaling will lead to radioprotective signals.1 Ionizing radiation produces complex multiple, and potentially lethal, DNA double strand breaks by direct energy deposition or generation of reactive oxygen species (Figure 2). The main DNA double strand break repair mechanisms make use of homologous recombination or nonhomologous endjoining (NHEJ). Homologous recombination takes place during S- and G2-phase of the cell cycle and is responsible for repair of approximately 20% of all DNA double strand breaks.22
The more error prone repair by NHEJ is responsible for repair of the majority of DNA double strand breaks caused by irradiation and is therefore potentially more important for treatment outcome.22,23 EGFR/PI3-K/AKT signaling is directly involved in the activation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) regulating DNA double strand break repair by NHEJ in K-RAS mutated cells and blockage of this pathway leads to significant impairment of DNA repair.24 EGFR activation, in a ligand independent manner or through up-regulation of autocrine ligands, is responsible for EGFR internalization. This is followed by nuclear translocation of EGFR together with DNA-PKcs which leads to an increase of the DNA-PKcs dependent NHEJ.25 Das et al. 26 showed that DNA-PKcs plays a critical role in EGFR-mediated radioresistance: in DNA-PKcs deficient cells the radioprotective effect is lost. Somatic mutations in the TK domain of EGFR in NSCLC cell lines significantly delayed DNA double strand break repair and reduced clonogenic survival in response to radiation by preventing nuclear transport of EGFR and/or association with DNA-PKcs.26,27 Targeting of EGFR and downstream pathways by either the specific TK inhibitor BIBX1382BS (BIBX) or the PI3-K inhibitor LY294002 enhanced radiation sensitivity in K-RAS mutated human tumor cells.28,29 EGFR inhibition by cetuximab, an IgG1 monoclonal antibody against the ligand-binding domain of the EGFR, can modulate the balance between cytoplasmic and nuclear DNA-PKcs and thereby reduces DNA repair.30,31
Activated RAS signaling to the PI3-K/AKT pathway leads to prolonged tumor cell survival after DNA damage has been inflicted, and hence radioresistance. This is irrespective whether RAS activation results from mutation of RAS or mutation or overexpression of receptor TK. This was illustrated by selective inhibition of either PI3-K or AKT in tumor cells with active RAS signaling resulting in increased radiosensitivity.32 In the majority of NSCLC cell lines AKT was constitutively active, independent of histologic subtype, and p53, Rb, or K-RAS status. Inhibition of AKT activity in these tumor cell lines by pharmacological or genetic approaches resulted in enhanced cellular responsiveness to chemotherapy and irradiation.11 RAS activation by mutation or by receptor TK activity is a frequent event in human tumors indicating that PI3-K/AKT mediated DNA damage repair pathway plays an important role in radioresistance in the clinical situation.
The PI3-K/AKT Pathway and Tumor Cell Proliferation
Tumor cell proliferation is influenced by several factors such as differentiation status, cell cycle gene regulation, and microenvironmental factors including oxygen and nutrient availability. Under hypoxic conditions progression of the tumor cell through the cell cycle is delayed, which may allow rapid repopulation under improved reoxygenated conditions.33 PI3-K/AKT is mainly a survival pathway but involvement of the PI3-K/AKT pathway in tumor cell proliferation was shown by its signaling to cyclin d-dependent kinases which regulate the cell-cycle G1/S phase transition.34 Preclinical data showed that G1 arrest as a result of inhibition of the EGF receptor prevented repopulation during radiotherapy.35
Accelerated tumor cell proliferation as a response to fractionated radiotherapy is linked to EGFR in a ligand independent manner. Irradiation leads to EGFR tyrosine phosphorylation, irrespective of the levels of EGFR expression, and through PIP3 activation of downstream pathways such as the RAF/Mitogen-activated protein kinase pathway to subsequent enhanced proliferation.36
Alternately, the inducible expression of a dominant negative EGFR led to decreased proliferation of tumor cells in response to irradiation. Combined inhibition of EGFR and ERBB2 further decreased AKT activity and proliferation in response to radiation.37
Another mechanism to influence response to radiation is combination treatment with the monoclonal antibody cetuximab. Inhibition of EGFR with cetuximab in human lung cancer cell lines and NSCLC xenografts showed marked inhibition of tumor growth when cetuximab was combined with radiation which can be explained by a shift to the G0/G1 phase of the cell cycle and reduced EGF induced phosphorylation of EGFR and ERBB2.38 In FaDu cells (human hypopharyngeal squamous cell carcinoma line) fractionated irradiation combined with cetuximab decreased repopulation and improved reoxygenation both contributing to local control. Underlying mechanisms for this improved reoxygenation may be rapid tumor regression and reduced oxygen consumption.39 These data provide a rationale for clinical studies to investigate the effect of combinating cetuximab with radiation in NSCLC.
The PI3-K/AKT Pathway and Hypoxia
Hypoxia is an important phenomenon in solid tumors leading to genetic instability and creation of more aggressive phenotypes causing resistance to chemotherapy and radiotherapy.
Cancer cells have the ability to undergo genetic and adaptive changes in response to hypoxia that allow them to survive and even proliferate under hypoxic conditions. HIF-1 is a key transcription factor induced by hypoxia which modulates expression of genes and their protein products involved in tumor growth and apoptosis.40,41 Upon activation HIF-1 leads to up-regulation of many gene products, including VEGF, the glucose transporters (GLUT-1 and GLUT-3) and carbonic anhydrase IX.42 Under normoxic conditions HIF-1α is rapidly deactivated by binding to the von Hippel Lindau protein or by factor inhibiting HIF-1 preventing further transcription. However, under hypoxic conditions transcriptional activity is regulated through the PI3-K/AKT pathway.41 Activation of this pathway results in increased transcription and expression of HIF-1. This is illustrated in human prostate cancer cells: HIF-1 transcription was blocked in the absence of AKT or PI3-K, and stimulated by constitutively active AKT or in dominant negative PTEN.43 Also in breast cancer cell lines, interruption of the PI3-K/AKT pathway by the selective PI3-K inhibitor, LY294002, inhibited HIF-1α induction resulting in a reduction of VEGF expression by 50%.44 The close interaction between hypoxia, angiogenesis, and radiosensitivity was shown in lung carcinoma xenografts. In these tumors AKT signaling was inhibited by the protease inhibitor nelfinavir resulting in a decrease of both VEGF and HIF-1α expression. This led to both a reduction of angiogenesis and a decrease in hypoxia in response to radiation.45
The PI3-K/AKT Pathway, Hypoxia, and VEGF
One of the important mechanisms by which EGFR inhibition enhances tumor oxygenation is by VEGF regulation. VEGF is crucial in tumor induced endothelial cell proliferation and vascular permeability leading to neo-angiogenesis. Prevention of neo-angiogenesis can result in a normalization of the vasculature and improved perfusion leading to a reduction of tumor cell hypoxia. VEGF is one of the most widely studied hypoxia-inducible proteins.46 Two distinct pathways, one through HIF-1α translation and one HIF-independent, have been recognized by which VEGF expression is regulated, both involving PI3-K and AKT.45,47 VEGF is one of the genes under control of HIF-1α in hypoxic conditions but it is also activated in normoxic conditions through the PI3-K/AKT pathway by EGFR or loss of PTEN.42,48 The EGFR/PI3-K/AKT pathway can control expression of VEGF either in a paracrine manner through EGF or through downstream signaling through the PI3-K/AKT pathway. In vitro and in vivo experiments showed that PI3-K inhibition leads to reduced VEGF expression. Also, pAKT counteracts the down-regulation of VEGF activity by TK inhibition indicating that PI3-K/AKT operates immediately downstream of the TK receptor controlling VEGF expression.49
Morelli et al.50 showed that angiogenesis can be decreased by VEGF-A blocking through EGFR inhibition. Cell growth was significantly inhibited by small molecule TK inhibition (gefitinib), by anti-EGFR blocking monoclonal antibodies (cetuximab) and by vandetanib, a tyrosine kinase inhibitor (TKI) that inhibits both VEGFR-2 and EGFR. This growth inhibiting effect was more pronounced when combinations were used of TKIs (gefitinib or vandetanib) and cetuximab that could be explained by effects on EGFR and downstream signaling to proliferation and survival pathways. The combination of cetuximab and vandetanib resulted in an almost complete suppression of phosphorylated EGFR, 50 to 70% reduction in EGFR protein levels and almost complete suppression of pAKT and mitogen-activated protein kinase. VEGF blockage through EGFR-inhibition in xenografts of human non-small cell lung adenocarcinoma (A549), significantly reduced angiogenesis. Microvessel density was decreased both by cetuximab and vandetanib and combined, the two agents led to a significant decrease in angiogenesis.50 This study showed that an optimal inhibition of EGFR can be obtained with concomitant inhibition of the extracellular ligand binding domain and the intracellular TK domain. Furthermore, an enhanced and more persistent control of tumor cell proliferation and angiogenesis was achieved by the combined inhibition of the two distinct but related signaling pathways (the EGFR and the VEGFR pathway).50 These findings highlight the close relation of EGFR and VEGF and downstream signaling to the PI3-K/AKT pathway.
CLINICAL RELEVANCE OF PI3-K/AKT ACTIVATION IN NON-SMALL CELL LUNG CANCER
EGFR and PI3-K/AKT Activation
EGFR is expressed in a variety of solid tumors including breast, prostate, colorectal, head and neck, and NSCLC. EGFR amplification occurs in up to 80% of NSCLC patients and initial reports suggested a poor prognosis. However, a meta-analysis failed to identify any association between EGFR expression and survival in lung cancer patients.51 Only a minority of EGFR positive tumors demonstrate a clinically meaningful response to EGFR inhibition.52–54
To increase response, targeting EGFR in combination with radiotherapy seems an attractive therapeutic strategy. The TKI erlotinib has shown to modulate response to radiotherapy in human NSCLC both in vitro and in vivo. Erlotinib enhanced radiation induced apoptosis, inhibited radiation induced activation of the EGFR receptor and attenuated radiation induced expression of RAD51, a measure of DNA double strand break repair. Tumor response of NSCLC xenografts to radiotherapy was improved when combined with erlotinib.55 Gefitinib also enhanced the cytotoxic effects of radiotherapy in NSCLC cell lines. Cellular repair of radiation-induced DNA double strand breaks was suppressed at pharmacologically achievable levels.56 This underlines the strong involvement of the EGFR/PI3-K/AKT signaling pathway regulating DNA double strand break repair through DNA-PKcs. In NSCLC preclinical data suggest a synergistic effect for cetuximab or EGFR TK inhibitors in combination with radiotherapy.55–59 In unresectable stage III NSCLC the role of cetuximab in combination with chemoradiotherapy is currently evaluated by the Radiation Therapy Oncology Group, by the continuing Cancer and Leukemia Group B 30407 trial and others.58,60
For cancer cells to be successfully inhibited by anti-EGFR, EGFR must be a critical driving force for its growth and survival and thus control PI3-K activity. Potential mechanisms for lack of response to EGFR-inhibition include constitutive activation of signaling pathways independent of EGFR and EGFR-independent activation of signaling pathways through a number of stimuli such as hypoxic stress, RAS-activation or PTEN-inhibition.38,48 In addition, the potential of cells to develop resistance to TK inhibition was shown by Engelman et al.61 In this study resistance was caused by amplification of the MET proto-oncogene, not through EGFR but through ERBB3 stimulation of the PI3-K pathway. Inhibition of AKT phosphorylation with LY294002 and wortmannin (AKT inhibitor) increased apoptosis but only in cells with activated AKT (pAKT). In cells with high pAKT, inhibition of pAKT in combination with either chemotherapy or irradiation led to increased apoptosis. Modulation of pAKT activity has shown to alter cellular responsiveness to chemotherapy and irradiation in NSCLC and offers opportunities for further research in clinical studies in patients with high levels of pAKT.11 Therefore, molecular predictors of sensitivity to EGFR directed therapy should not only assess the EGFR status but need to be extended to quantify the EGFR-activation status and key markers of the activated downstream signaling pathways.62
PI3-K/AKT Activation as a Prognostic Factor in Non-small Cell Lung Cancer
Preclinical data showed pAKT positivity, using immunoblotting, in 80% of NSCLC cell lines independent of tumor histology.11 Clinical data using immunohistochemical staining to measure activated pAKT in pretreatment tumor specimens varied from 50 (stage I–III) to 84% (stage III) of patients.17,63 Importantly, there was a discrepancy between pAKT expression and EGFR expression.17 These findings are consistent with previous findings in head and neck cancers.18 This suggests that, in the clinical situation, EGFR-independent activation or down-regulation of the PI3-K/AKT pathway could be a frequent event (Figure 3).
In patients who underwent surgical resection for NSCLC with lymph node metastasis, pAKT correlated with poor prognosis (5-year survival roughly 20 versus 60% for pAKT negative patients). No association was found with other clinical characteristics.64 Although in this study survival data may be confounded because of a lack of consistence for adjuvant therapy it clearly shows the clinical importance of activated signaling through the AKT pathway and its impact on treatment outcome and prognosis.
PI3-K/AKT Activation as a Target for Treatment in Non-small Cell Lung Cancer
In vitro studies with NSCLC cell lines showed synergistic effects if multiple cell signaling proteins are targeted simultaneously.65 Persistent activity of the PI3-K/AKT or RAS/ERK-pathway, indicated by elevated levels of phosphorylated AKT (pAKT) or ERK (pERK), despite EGFR-TK-inhibition by gefitinib indicated treatment resistance. Cells with persistent activity of the PI3-K/AKT or RAS/ERK-pathways after EGFR-TK-inhibition were treated in combination with specific inhibitors of PI3-K (Ly294002) or RAS (farnesyl transferase inhibitor SCH66336), respectively. The combination of EGFR inhibition and PI3-K/AKT or RAS/ERK-inhibition clearly showed additive cytotoxicity.65 Therefore, it is likely that EGFR-TK-inhibitors, such as gefitinib, in combination with specific inhibitors of down stream signaling pathways involved in cell survival, such as the PI3-K/AKT or RAS/ERK-pathway, is a promising strategy to overcome treatment resistance in NSCLC.
In NSCLC cells lines with high levels of pAKT, pharmacological inhibiting of P13-K led to decreased AKT phosphorylation resulting in radiosensitization and an increase in chemotherapy induced apoptosis.11,17 Inhibition of the PI3-K/AKT pathway can also be achieved by use of protease inhibitors. Treatment with these compounds resulted in a decrease in pAKT in various cell lines, including NSCLC, leading to increased radiosensitivity. In addition, overexpression of active PI3-K in cells without activated AKT resulted in radiation resistance that could be inhibited with protease inhibitors. Finally, in xenografted tumors down-regulation of pAKT was seen at a dose range comparable with the therapeutic levels used in HIV patients and a synergistic radiosensitisation was observed when nelfinavir was combined with radiotherapy.49,66 These studies emphasize the potential for specific targeting the activated PI3-K/AKT pathway to counteract radiation induced cellular defense mechanisms.
It is clear that P13-K/AKT is an important regulator of various cellular functions including proliferation, invasion, apoptosis, and up-regulation of hypoxia-related proteins. Consequently, this pathway plays a key role in the three major radiotherapy resistance mechanisms, also in NSCLC: intrinsic radiosensitivity, tumor proliferation, and tumor cell hypoxia. Expression of pAKT is an independent prognostic indicator of clinical outcome of lung cancer in retrospective studies. Blocking of the PI3-K/AKT signaling routes through EGFR-inhibition improves radiosensitivity and can affect tumor growth substantially through various mechanisms including inhibition of neo-angiogenesis via VEGF. Importantly, EGFR-independent activation of the PI3-K/AKT pathway commonly occurs and is an important factor in treatment resistance. Prospective studies are needed to quantify the activated status of the EGFR/P13-K/AKT pathway and may be helpful in selecting patients for treatments combining chemotherapy and radiotherapy with targeted therapy.
The Dutch Cancer Society, grant number 2008-4000 provided financial support.
We thank J.P.W. Peters for technical support.
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