Molecular targeting in radiotherapy of lung cancer

Lung Cancer 45 Suppl. 2 (2004) S187–S197

www.elsevier.com/locate/lungcan

Molecular targeting in radiotherapy of lung cancer Michael Baumann a,b, *, Mechthild Krause a , Daniel Zips a , orr a,b , Cordula Petersen a , Klaus Dittmann c , Wolfgang D¨ Hans-Peter Rodemann c a

Dept. of Radiation Oncology, b Experimental Center, Medical Faculty and University Hospital Carl Gustav Carus, University of Technology, Dresden, Germany c Section of Radiobiology & Molecular Environmental Research, Dept. of Radiation Oncology, Eberhard-Karls University, T¨ ubingen, Germany

KEYWORDS Radiotherapy; Tumour response; Normal tissue reactions; Molecular targeting; EGFR; COX-2; Angiogenesis; KGF; TGF-b; BBI; Translational research; Tumour growth delay; Local tumour control

Summary Molecular targeting is a promising option to increase the radiation response of tumours and to decrease normal tissue reactions, i.e. to achieve therapeutic gain. Molecular targeting substances in themselves are not curative while radiation is a highly efficient cytotoxic agent, with local recurrences often occurring from only few surviving clonogenic cells. High-dose radiotherapy therefore offers optimal conditions to evaluate the potential of specific biology-driven drugs for oncology. This review summarises the current status of preclinical and clinical research on combined radiation with examples of molecular targeting substances relevant for the treatment of NSCLC (EGFR, COX-2, VEGFR, KGF, TGF-b, BBI). © 2004 Elsevier Science Ltd.

1. Introduction Lung cancer is the leading cause of cancer death in industrialised countries [1,2]. Histologically about 80% of all lung cancers are non-small-cell lung cancers (NSCLC) [3]. Surgery is the mainstay of therapy in stage-I and -II disease, and for selected patients with locally advanced stage-III tumours [4]. Postoperative radiotherapy may significantly im* Correspondence to: Prof. Dr. M. Baumann. Dept. of Radiation Oncology, Medical Faculty Carl Gustav Carus, University of Technology Dresden, Fetscherstr. 74, D-01307 Dresden, Germany. Tel.: +49-(351)-458-2095; fax: +49-(351)-458-5716. E-mail: [email protected]

prove local tumour control in completely resected N2 disease and after R1/2 resections and is often recommended in these situations [5]. Unfortunately only about 30% of all patients with NSCLC fall in the group of operable tumours. If the tumour is unresectable, or if the patient is inoperable, radiotherapy given either alone or combined with chemotherapy offers the only curative chance [5,6]. For several decades conventional fractionation, i.e. the application of 1.8–2 Gy per fraction in 5 fractions per week to total doses of 60–70 Gy has been the gold standard of radical radiotherapy for NSCLC [4,6,7]. Large randomised clinical trials performed in the last years have demonstrated

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S188 a modest but clinically relevant improvement of outcome by combined radiochemotherapy or by highly accelerated and hyperfractionated (CHART) radiotherapy [6,8–11]. Therefore one of these treatments should today be considered as best care of patients with NSCLC. However, even if state-of-the-art radiotherapy or radiochemotherapy is applied in inoperable NSCLC about 80% of all patients will eventually develop a local recurrence and about 60% distant metastases [5,6]. These disappointing figures call for further improvement of both local regional as well as systemic therapy.

2. Aim of curative radiotherapy The aim of curative radiotherapy in NSCLC is to inactivate all clonogenic cells in the primary tumour as well as in hilar and mediastinal lymph nodes without producing unacceptable damage in surrounding normal tissues, most importantly lung. It is well recognised from experimental and clinical data that local tumour control increases with increasing total radiation dose [4,12,13]. In the steep part of the dose–response curve an increase of dose by 10% may increase local tumour control in a mixed population of tumours of the same histology and stage by approximately 10–20% [13]. Thus, escalation of the total dose from 60 to 70 Gy to doses of, say, 100 Gy should theoretically result in a major improvement of local tumour control and thereby also in a significant improvement of survival. However not only tumour control but also the incidence and severity of normal-tissue damage increases with increasing total doses of irradiation [12,14]. Radiation dose–response curves for normal-tissue damage are usually very steep while, because of intertumoral heterogeneity, dose–response curves for tumours tend to be flatter [4,13]. Structural damage of the lung after irradiation, i.e. the degree of pneumonitis and fibrosis per unit of tissue, depends mainly on the biological equivalent dose which is a function of total dose, dose per fraction and, to a lesser extent and only for pneumonitis, overall treatment time [4,15,16]. In contrast, functional lung damage shows a pronounced volume effect, i.e. it depends on the proportion of lung treated to doses above the threshold for structural damage [17,18]. From these radiobiological considerations it can be concluded that simplistic escalation of total radiation dose in NSCLC without well-defined volume restrictions for irradiation of normal lung may result in a reduced rate of uncomplicated local tumour control, i.e. a decreased therapeutic ratio. Such an effect was observed in the randomised RTOG 83-11 phase-I–II dose escalation trial on hyperfractionated

M. Baumann et al. radiotherapy [19]. The results of a subgroup analysis of this trial suggest an improved outcome with increasing radiation dose between 60 and 69.6 Gy but not after doses higher than this. The incidence of severe radiation-induced pneumopathy increased from 2.6% after 60–64.8 Gy to 5.7% after 69.6–74.4 Gy and 8.1% after 79.2 Gy. Thus, increased morbidity might have contributed to the lack of a positive dose–response relationship at doses higher than 69.6 Gy. This is in line with the conclusion from the classical work of Holthusen [12], that once the optimum dose for a given disease, treatment schedule and radiation technique has been established, further improvement of uncomplicated local tumour control can only be achieved by either moving the dose–effect curve for local tumour control to lower doses or the curve for normal-tissue damage to higher doses.

3. Rationale for molecular targeting in radiotherapy Until recently, clonogenic tumour cells were considered to be the only target of radiotherapy. Inactivation of clonogenic cells by radiation was thought to be a stochastic process that is independent of interactions between tumour cells and between tumour cells and other tissues, e.g. intratumoral stroma. It was well recognised that the radiosensitivity of tumour cells may vary substantially between different tumours (intertumoral heterogeneity) but also, for example as a function of the oxygenation status and the cellcycle distribution, between the clonogenic cells within one tumour (intratumoral heterogeneity). Many investigations clearly support the importance of the direct inactivation of tumour cells by radiation for local tumour control and show that, under well-defined experimental conditions, cell kill in tumours is an exponential function of radiation dose [20,25]. While the primary focus of cancer research on the clonogenic tumour cells has been termed “reductionist view” in a recent seminal review by Hanahan and Weinberg [26], it is of enormous scientific value for radiobiology and radiotherapy and continues to be the quantitative basis for investigating the efficacy of novel treatment strategies. Having said this, it must also be clearly stated that complex interactions do exist between the different cells and tissues in tumours and that this so-called “heterotopic cell biology” [26] opens novel strategies to improve the outcome of radiotherapy. In fact, radiotherapy might eventually be an optimal modality to integrate molecular targeting approaches.

Molecular targeting in radiotherapy of lung cancer Firstly, the radiobiological mechanisms of the response of tumours and normal tissues to radiotherapy are well recognised (so called Rs of fractionated radiotherapy [27]). The molecular mechanisms that underlie these Rs, and the molecular consequences of the Rs are increasingly known [28]. Many of the molecular target molecules are differently expressed between tumours and normal tissues. This offers the possibility of specific, biology-driven modulation of the radiation response in tumours and normal tissues, and thereby a therapeutic gain. Secondly, similarly as conventional chemotherapeutic drugs, almost all of the novel therapeutic agents developed so far are in themselves not curative, especially for solid tumours. In contrast, radiotherapy in itself is extremely efficient in eradicating clonogenic cells, and recurrences often occur from only one or a few surviving cells [29]. Thus, even if novel biologically targeted drugs are only able to kill a limited number of clonogenic tumour cells, this might be sufficient to increase local control when combined with radiotherapy. The same argument applies when these drugs increase the radiosensitivity of tumour cells, or when normal tissues are specifically protected and the radiation dose to the tumour can be escalated. Thirdly, in contrast to systemic chemotherapy, radiotherapy cannot only be modified in dose and time but also in space. This allows individual tailoring of the effects of combined treatments under consideration of the spatial distribution of clonogenic tumour cell burden as well as under consideration of sparing of normal tissues. From this reasoning, the combination of radiotherapy or radiochemotherapy with molecular targeted drugs appears a highly promising avenue for preclinical and translational cancer research [30,31]. Some examples of combinations that may improve tumour response or may protect normal tissues will be discussed in the following.

4. Tumour-specific enhancement of radiation effects Enhancement of the effect of radiation on tumours by molecular targeted drugs exploits specific characteristics, which are acquired by tumour tissue in the process of carcinogenesis and tumour progression. Independent of whether the drug targets the tumour cells directly or the tumour cells are affected indirectly, e.g. by antiangiogenic drugs, the efficacy of the treatment depends on inactivation of clonogenic cells (compare sect. 3). By definition, the last tumour cell which is able

S189 to produce a recurrence (so-called tumour rescuing unit or clonogenic tumour cell) must be killed to achieve permanent local tumour control. An important problem in cancer research is that tumours contain a mixture of clonogenic and nonclonogenic tumour cells. These populations can be discriminated only functionally on the basis of their capacity to produce an expanding number of daughter cells, i.e. to form a recurrence. Tumour growth delay after subcurative doses, which is used in many preclinical studies, depends on the inactivation and regrowth rate of both clonogenic and non-clonogenic cells. In contrast, determination of radiation dose–effect curves for local tumour control depends solely on the inactivation of clonogenic cells [29,32]. It therefore appears preferable to the authors to use local tumour control as experimental endpoint when the effect of combined irradiation with molecular targeting substances on survival of clonogenic tumour cells is investigated [33]. Alternatively, large growth-delay studies that carefully evaluate several dose levels, and that correct for different growth rates in different experimental arms [34], may yield results as reliable and as relevant as tumour control assays [33,35]. The following subsections summarise the status of research on three groups of modulators of radiation effects on tumours, two of them with direct activity against tumour cells (EGFR and COX-2 inhibitors), and one with indirect activity via inhibition of tumour angiogenesis. 4.1. Inhibition of the epidermal growth factor receptor The epidermal growth factor receptor (EGFR/ ErbB1/HER1) is a member of the ErbB tyrosinekinase receptor family. Activation of the receptor by EGF-like ligands or irradiation results in cell proliferation, angiogenesis, and radioresistance [36,37]. EGFR is overexpressed in a variety of solid human tumours including NSCLC, head and neck squamous cell carcinomas and glioblastomas. Overexpression of the EGFR is associated with poor prognosis and radioresistance of tumours [38,41]. Therefore, inhibition of EGFR in combination with irradiation bears promise for cancer therapy [42]. In most studies testing this strategy monoclonal antibodies (mAbs) and tyrosine-kinase (TK) inhibitors have been applied [32,43,50]; however, genetherapeutic approaches were also used [51]. Monoclonal antibodies target the extracellular domain of EGFR and prevent homo- or hetero-dimerisation and thereby activation of the receptor. Tyrosinekinase inhibitors are small molecules, which block

S190 EGFR signal transduction by inhibiting autophosphorylation of the intracellular tyrosine kinase and the subsequent phosphorylation of target molecules. Preclinical studies have shown inhibition of tumour cell proliferation and radiosensitisation in vitro [43,46] as well as prolongation of tumour growth delay after combined EGFR inhibition with mAb or TK inhibitors and irradiation in vivo [32,47,50]. So far only few authors have investigated local tumour control after such combined treatment. While tumour growth delay depends on the inactivation and regrowth rate of both clonogenic and non-clonogenic cells, local tumour control reflects inactivation of clonogenic cells and therefore is the preferable endpoint for experimental radiotherapy. After single-dose irradiation and application of the anti-EGFR antibody C225 local tumour control was improved in the highly EGFR-overexpressing A431 squamous cell carcinoma. This improvement was significant after 3 injections of the mAb (6 h before, 3 d after and 6 d after irradiation) but not after a single application of C225 6 h before irradiation [52]. After fractionated irradiation with 30 fractions in 6 weeks and simultaneous EGFR-TK inhibition, growth delay of FaDu squamous cell carcinoma was significantly prolonged but local tumour control was not improved [32]. The same result was obtained after adjuvant EGFR inhibition after fractionated irradiation in the same tumour model [53]. So far no randomised trials are available that investigate the effect of EGFR inhibitors in combination with radiotherapy in NSCLC. However, the results of several clinical studies are helpful in discussing potential and problems of this approach. In a clinical phase-I study on patients with advanced NSCLC, ovarian, head and neck, prostate or colorectal cancer, application of the EGFRTK inhibitor ZD1839 (Iressa) resulted in a stable disease in 19/88 patients with tolerable side effects [54]. The IDEAL 1+2 trials showed overall response rates of 9–19% when the EGFR-TK inhibitor gefitinib (Iressa) was applied in stage-III–IV NSCLC which failed after chemotherapy [55,57]. However, when gefitinib was combined in the INTACT 1+2 trials with polychemotherapy in chemotherapynaïve NSCLC, neither the overall response rate nor survival was improved compared to chemotherapy plus placebo [57,59]. Several possible explanations for these unexpected results have been offered, including imbalances of tumours with different EGFR expression (which were not determined in these trials), targeting of the same subpopulation of tumour cells by chemotherapy and gefitinib, and deletion of the effect of EGFR inhibitor by effective chemotherapy [57]. Several phase-I–III

M. Baumann et al. trials testing the effect of different small molecule EGFR inhibitors combined with radiochemotherapy in NSCLC are currently ongoing [60,61]. In head and neck squamous cell carcinoma inhibitors of the EGFR have been applied simultaneous with radiotherapy. In a phase-I study on 15 patients, 13 complete and 2 partial remissions were observed after simultaneous C225 mAb and radiotherapy. The 2-year disease-free survival was 65%. At a loading dose of 400–500 mg/m2 and a maintenance weekly dose of 250 mg/m2 the mAb was well tolerated [62]. A phase-III study on C225 plus radiotherapy has completed accrual, however, results are not available yet [63]. From the preclinical and clinical data it can be concluded that inhibition of EGFR decreases the growth rate of tumours expressing this receptor. However, when combined with radiation or chemotherapy, treatment outcome is not always improved by EGFR inhibitors. Of special importance for radiotherapy is that improved growth delay after combined EGFR inhibition (simultaneous and adjuvant) with fractionated radiation does not necessarily improve local tumour control, which is by far the most important endpoint for this treatment modality. This observation underscores the need for specific preclinical experiments on combined treatments of molecular targeting drugs with radiation using clinically relevant treatment schedules and local tumour control as endpoint. Extrapolations on the efficacy of combined treatments with radiation based on growth delay after EGFR inhibition alone or in combination with chemotherapy are not sufficient. Furthermore, experimental data indicate heterogeneity of the response to EGFR inhibition between different EGFR-positive tumours. For example, Toulany et al. [64] have shown that, as a consequence of the specific mutation pattern, EGFR inhibition in some tumours mainly reduces tumour cell proliferation, whereas in other tumours little effect on proliferation but pronounced radiosensitisation was observed. Therefore further research exploring the mechanisms of EGFR inhibition in the context of radiotherapy are needed to exploit the potential of such combinations and to develop predictive tests that direct their use. 4.2. Inhibition of COX-2: Targeting the prostanoid pathway The rate-limiting enzyme in the synthesis of prostaglandins (PGs) is cyclo-oxygenase (COX). Since 1991 it is known that two isoforms of cyclooxygenase exist: COX-1 and COX-2 [65,66]. Whereas COX-1 is constitutively expressed in most tissues and mediates the synthesis of PGs required for

Molecular targeting in radiotherapy of lung cancer normal physiological functions, COX-2 is typically not expressed or expressed at relatively low levels in undisturbed healthy tissues, but is inducible by an assortment of agents including pro-inflammatory stimuli, mitogens and/or hormones depending on the tissue [66]. COX-2 is overexpressed in 40–80% of cancers of the lung, colon, head and neck, breast, prostate, brain and pancreas [67,69]. On the basis of the cytoprotective role of prostaglandins against irradiation [69,70], non-selective COX inhibitors, such as indomethacin, have been shown to enhance tumour radiation response in vitro [71,72]. More recently, selective COX-2 inhibitors are favoured because of their improved toxicity profile on the gut epithelium. In vitro, the COX-2 inhibitors SC-236 and NS-398 exhibit additive effect to radiation in human glioma and lung cancer cells, respectively [73,74]. However, in nude mouse models of rodent and human cancers, SC-236 confers supraadditivity to the tumour growth-inhibitory effect of irradiation or SC-236 alone [73,75,76]. Although complete tumour regression and improved radiocurability were reported after single irradiation doses of 30–50 Gy, these doses are not directly applicable to fractionated clinical radiotherapy protocols [75]. Toxicity to normal tissues was not excessive in these experimental studies. Augmentation of tumour response to radiotherapy by COX-2 inhibitors seems to be dependent on the presence of tumoral COX-2 expression [74]. The mechanism is unclear, and some groups suggest that it might be related to enhancement of irradiationinduced apoptosis [74,75] although this hypothesis has been refuted by other studies [73,76]. Other possibilities include the modulation of tumourintrinsic radiosensitivity [73] and tumour angiogenesis [75,76]. Clinical trials combining COX-2 inhibitors and radiotherapy are feasible and ongoing. Six clinical trials evaluating combined modality therapy with the selective COX-2 inhibitor celecoxib have been initiated in NSCLC [61]. RTOG is conducting two of these trials. One is a phase-II postoperative adjuvant study of 2 years of celecoxib (400 mg bid) and radiation (50.4 Gy) in completely resected stage-I/II NSCLC patients. The second is a phase-I/II trial in locally advanced NSCLC. Patients are treated with fractionated irradiation (66 Gy) and twicedaily celecoxib for up to 2 years. In both studies it will be determined whether celecoxib improves response and survival. Clinical trials combining celecoxib, chemotherapy and radiotherapy are also being conducted in other cancers in which radiation is a prominent component of standard therapy, i.e. in patients with unresectable or potentially resectable oesophageal cancer, in patients with

S191 locally advanced cancer of the cervix, and in patients with glioblastoma multiforme [77]. The results of these early-phase studies, which are expected over the next few years, will determine whether the current enthusiasm for combining COX-2 inhibitors and radiation is maintained and justifies the initiation of randomised controlled clinical trials. 4.3. Inhibitors of tumour angiogenesis / Antiangiogenic substances Formation of new blood vessels, i.e. angiogenesis, is an indispensable requirement for development and progression of malignant tumours and metastasis [78,80]. The complex cascade of angiogenesis includes angiogenic stimulation of endothelial cells, degradation of basement membrane, migration and proliferation of endothelial cells and subsequently differentiation into a three-dimensional supplying vascular network. Each step is tightly regulated by numerous endogenous promoters and inhibitors of angiogenesis. These molecules involved in angiogenesis represent targets for rationally designed inhibitors of tumour angiogenesis. Angiogenic factors such as VEGF are highly expressed in solid tumours including lung cancer and are related to poor clinical outcome [81,82]. Extensive experimental data indicate that either inhibitors of pro-angiogenic factors or administration of endogenous anti-angiogenic factors reduce the formation of new blood vessels. As a result, tumours grow at a slower rate or even shrink. However in most cases no permanent tumour control can be achieved. In pre-clinical studies anti-angiogenic agents combined with irradiation have been shown to prolong tumour growth delay and increase local tumour control compared to either treatment alone. Several mechanisms have been suggested to explain the radiation-response-modifying effect of anti-angiogenic agents, including an improved tumour oxygenation, decreased vessel density, increased tumour cell loss, reduced tumour cell proliferation, and radiosensitisation of endothelial cells [83,85]. In the majority of experimental studies anti-angiogenic agents were given concurrently with irradiation in order to take advantage of the radiosensitising effect. However, experiments comparing different combination schedules of antiangiogenic compounds and irradiation also have shown a benefit for adjuvant administration of inhibitors of angiogenesis [86]. A major concern of the combination of antiangiogenic approaches with irradiation is that tumour hypoxia might increase and thereby radiosensitivity of tumour cells decrease. Such

S192 a negative effect of anti-angiogenic agents on the results of irradiation was observed after administration of TNP-470 and suramin [87,88]. Yet, this observation was not confirmed when other anti-angiogenic agents, e.g. VEGF inhibitors, were administered [89,90]. Moreover, some studies imply that VEGF inhibition may even increase tumour oxygenation by selective ablation of immature tumour blood vessels, a decrease in the number of oxygen-consuming cells and a decrease in vessel permeability [91]. A number of different anti-angiogenic drugs have been tested in clinical trials in the treatment of lung cancer, alone or in combination with chemotherapy [92,93]. In a clinical Phase-I trial angiostatin, an endogenous inhibitor of angiogenesis, was given concomitant with fractionated radiotherapy for solid tumours including lung cancer. A partial tumour response in all 13 evaluable patients but no enhancement of radiation treatment toxicity, e.g. mucosal reactions, was observed [94]. So far no data are available from randomised clinical trials addressing whether antiangiogenic compounds can improve the outcome after radiotherapy in NSCLC. However, several randomised Phase-III clinical trials are underway (www.cancer.gov). For example, Neovastat (AE-941), a naturally occurring agent with activity against multiple angiogenic targets, is currently under investigation in a randomised, double-blind, placebo-controlled, multicentre study. Patients with NSCLC stage III are treated with a radical radiotherapy plus neoadjuvant and concomitant chemotherapy. AE-941 (Neovastat) or placebo is administered twice daily beginning on day 1 or within 10 days of initiation of chemotherapy. Also, ECOG recently opened a Phase-III randomised study of carboplatin, paclitaxel, and chemoradiotherapy with or without Thalidomide, a compound that exerts immunomodulatory and antiangiogenic effects, in patients with stage-III NSCLC (www.ecog.org). In conclusion, pre-clinical and first clinical data suggest that anti-angiogenic agents can improve the results of radiotherapy. So far the use of anti-angiogenic compounds in radiotherapy is investigational and it will take years until their role in the treatment of lung cancer will be clarified.

5. Normal tissue-specific radioprotection Specific radioprotection of normal tissue represents a promising approach to improve radiotherapy by means of minimising normal-tissue responses. This

M. Baumann et al. may increase the therapeutic ratio by allowing either to increase the dose to the tumour and thereby local tumour control or to decrease normal-tissue effects in situations where local control rates are already high. Consequently, the ultimate criterion for categorising a substance as a normal-tissue selective radioprotector is the exclusion of tumour tissue from the protective effect. Radioprotectors of the current generation, such as amifostine, are not explicitly normaltissue specific [95]. Therefore the potential of such substances to improve the therapeutic ratio is limited. The following subsections summarise the status of research on three modulators of radiation effects with the potential of being almost specific (KGF, TGF-b) or completely specific (BBI) for normal tissues. 5.1. Application of keratinocyte growth factor (KGF) KGF is a heparin-binding growth factor that exerts effects on epithelial cells in a paracrine fashion through interaction with KGF receptors. This growth factor is known to interfere with various patho-physiological processes in lung tissue after injury or during inflammatory reactions [96,97]. These processes include proliferation and differentiation of type-II pneumocytes [98,99], surfactant production and homeostasis [99] as well as barrier function [100]. Protection against radiationor bleomycin-induced lung injury by KGF has been demonstrated in experimental models [101], when the growth factor was administered intratracheally. Recently, intravenous administration has also been shown to be effective [102]. These results suggest a potential role of systemic KGF administration for protection of radiation-induced lung damage, which, however, has to be tested in clinical trials. Amelioration of acute radiation effects in squamous epithelial tissues has extensively been demonstrated for mouse oral mucosa with single-dose as well as fractionated irradiation protocols [103,105]. Recently, in a double-blind placebo-controlled clinical phase-III trial of bone marrow ablation by total body irradiation and high-dose chemotherapy, significant reduction in oral mucositis was seen with recombinant human KGF [106]. Hence, as the epithelium of the oral cavity and the oesophagus are similar in morphological, physiological and radiobiological aspects, protection of acute radiation-induced oesophageal reactions, including dysphagia, may be expected from the administration of KGF. The promising results obtained so far warrant further systematic preclinical and clinical evaluation of the potential of KGF to prevent or

Molecular targeting in radiotherapy of lung cancer decrease dose-limiting normal-tissue reactions in the context of radiotherapy of NSCLC. These studies must address both normal-tissue protection and a possible reduction of the effect of radiotherapy on tumours, which cannot completely ruled out based on present data [104]. 5.2. Strategies against transforming growth factor beta (TGF-b1) Recent experimental and clinical data indicate that the cytokine TGF-b1 is a main modulator of early radiation-induced lung injury and late effects such as fibrosis. Based on its important role in regulating proliferation and differentiation through cell-cycle control mechanisms and to some extent also apoptosis, TGF-b1 offers an ideal target for antagonising the adverse effects of radiation on normal lung tissue. It has been shown recently that radiation exposure induces TGF-b1 gene expression in normal cells as well as activation of this cytokine [107,108]. Upon transcriptional activation TGF-b1 is produced in an inactive latent form as LTGF-b and secreted into the extracellular milieu, where it can bind in its latent form to extracellular matrix components like collagen [109]. Upon various stimuli LTGF can be activated proteolytically into TGF-b1 which is able to bind to the TGF-b1type II receptor (TbetaRII) which will then bind and activate the TGF-b1-type I receptor (TbetaRI) with a serine/threonine kinase activity. The protein-kinase activity of TbetaRI induces TGF-b1-dependent intracellular signaling through the phosphorylation of Smad-2 and Smad-3 proteins. These proteins will then form a complex with Smad-4, which is translocated to the nucleus and is necessary to transactivate TGF-b1-dependent gene expression, i.e. induction of p21Waf1, p27Kip, collagen, as well as TIMP [109,110]. The induction of these TGF-b1-dependent genes is most likely responsible for the cellular (modulation of proliferation and differentiation of fibroblasts) as well as biochemical (collagen deposition through enhanced synthesis and reduced degradation) events characteristic of radiation-induced late normal lung complication such as fibrosis. Consequently, targeting TGF-b1 by specific antagonists is a promising strategy to prevent radiation-induced normal-tissue damage during and after radiotherapy. As antagonistic tools in preclinical animal studies so far, TGF-b1 neutralising antibodies, soluble TbetaRII, as well as superoxide dismutase (SOD) have been used to prevent radiation-induced early and late normaltissue damage. Convincing results for preventing acute and late radiation-induced lung injury have been reported on the basis of animal experiments

S193 for soluble TbetaRII [111] as well as SOD [112]. Currently however, the application of these components requires gene-therapy approaches, which will not easily be deliverable in routine clinical radiotherapy. Consequently other strategies exploiting the development of specific and potent small-molecule inhibitors of TGF-b1-receptor kinase activity are currently under investigation. 5.3. Bowman–Birk protease inhibitor (BBI) The Bowman–Birk protease inhibitor (BBI), which has earlier been described to prevent in vitro and in vivo radiation-induced carcinogenesis [113], was found to be normal-tissue specific in in vitro cell culture experiments, applying normal fibroblasts and human tumour cell lines, as well as in ongoing animal studies. These investigations clearly show a significant protective effect during singleand fractionated-dose irradiation both on the basis of clonogenic assays in vitro [114,115] as well as the leg contracture assay in vivo [Dittmann et al. 2004, in preparation]. Investigations into the molecular mechanisms revealed that the radioprotective effect is dependent upon the presence of a functional wild-type tumour-suppressor protein p53 (TP53) and most likely involves an activation of the DNA repair machinery [115,116]. It has been shown that treatment with BBI prior to irradiation reduces the amount of chromosomal abnormalities as measured by the appearance of dicentric chromosomes, which is correlated with an enhanced activity of the DNA-PK, an enzyme engaged in the non-homologous end-joining repair mechanism of DNA doublestrand breaks [117]. The radioprotective activity of BBI is most likely dependent on a phosphotyrosine residue in its active center [118], and the application of the phosphorylated single amino-acid P-Tyr to human skin fibroblasts in culture revealed a similar radioprotective effect as the whole BBI molecule [119]. Since about 70% of all human tumours present a loss of TP53 function, it can be assumed that the clinical application of BBI or P-Tyr to protect normal tissue from radiation damage would effectively improve the therapeutic outcome of radiation therapy of p53-mutated tumours.

6. Conclusion Molecular targeting is a promising avenue to increase the radiation response of tumours and to decrease normal-tissue reactions, i.e. to achieve therapeutic gain. Molecular targeting substances in themselves are not curative. In contrast, radiation is a highly efficient cytotoxic agent, with local recurrences often occurring from only

S194 a few surviving clonogenic cells. The mechanisms mediating radioresistance are well understood on a radiobiological level and the molecular basis becomes increasingly known. High-dose radiotherapy therefore offers optimal conditions for evaluating the potential of specific biology-driven drugs for oncology. Such substances may act directly on tumour cells or indirectly via other cell compartments of the tumour, e.g. vasculature. The mode of action may be independent inactivation of clonogenic cells or modulation of the cellular response to radiation. So far a large body of studies in cell cultures and a variety of proof-of-principle experiments in simple in-vivo systems are available, while clinically relevant in vivo experiments and especially translational studies into the clinical setting are still rare. The most important overall conclusions and implications that can be drawn based on the present data are: (a) Proof-ofprinciple could be obtained for several approaches combining molecular targeting with radiation for both improved tumour response and decreased normal tissue reactions. (b) Most studies performed so far focus either on tumour response or on normal-tissue reactions, which hampers evaluation of therapeutic gain. (c) Many molecular targeting approaches decrease the growth rate of tumours when given alone or combined with radiation. This does not necessarily reflect enhanced kill of clonogenic tumour cells and improved curative potential. Specific radiotherapy-driven experiments using clinically relevant treatment schedules and endpoints are therefore necessary to further explore the potential of novel combinations; (d) Important heterogeneity of the efficacy of combined radiation–molecular-targeting approaches between different tumours necessitates further mechanistic research, use of several model systems in each step of translational research, and the development of predictive assays.

Acknowledgement Supported in part by the Deutsche Forschungsgemeinschaft (Ba 1433-2) and by the Deutsche Krebshilfe (T I/97/Ba I).

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