Radiotherapy and cellular signalling

Review

Radiotherapy and cellular signalling

Radiotherapy and cellular signalling Conrad R Lewanski and William J Gullick

Developments in cellular and molecular biology in the past decade have increased our understanding of the processes by which cells respond to ionising radiation. Cells use complex protein signalling systems that recognise radiation damage to DNA and plasma membrane lipids. When damage is found, it leads to the activation of various intracellular pathways that modulate the activity of genes controlling cellular responses such as apoptosis, cell-cycle arrest, or repair. Numerous molecular targets may be activated or inhibited in an attempt to upregulatre or downregulate the radiation response. In this review, we discuss some of the new compounds and techniques for manipulating the cell’s response to radiation. Lancet Oncol 2001; 2: 366–70

Since the discovery of ionising radiation in 1895, radiotherapy has gained a central therapeutic role and has become established as a first-line treatment in many human malignant diseases. Cell death from radiation is thought to result from damage to two cellular constituents – DNA and the plasma membrane. The damage is caused by ionisation of water inside cells, which produces damaging free radicals. It is estimated that each gray unit (1 Gy) of radiation produces 105 ionisations per cell, which cause about 2000 single-strand and 40 double-strand breaks in DNA, in addition to other types of DNA damage. Cell death from radiation correlates most closely with the number of doublestrand DNA breaks. However, many of the effects of ionisation are counteracted by free-radical-scavenging processes and cellular repair mechanisms. Until recently, the mechanisms by which cells recognise ionising radiation damage and respond to it were unknown, but now they are steadily emerging. Cells use complex protein signalling systems, which recognise radiationinduced oxidative damage to DNA and plasma membrane lipids, and stimulate intracellular signalling pathways. These pathways modulate the activity of genes controlling cellular responses such as apoptosis, cell-cycle arrest, or repair, and provide potentially attractive targets for pharmacological manipulation (Figure 1).

Cellular signalling pathways Ionising radiation induces an ever-expanding list of genes. Immediate-early genes encode transcription factors such as JUN and EGR1 (early growth response) that can bind to specific DNA sequences and modulate the expression of other genes. Although the reason why these early response genes are induced is unclear, they may help cells survive after radiation. Interference with normal EGR1 and JUN function in human epithelial cells using dominant negative constructs reduces survival after radiation.1 In addition, radiation induces a host of other intermediate and late366

Figure 1. Molecular developments in cell signalling will soon have impact on radiotherapeutic strategies. The stucture of ZD-1839 (Iressa), a new tyrosine kinase inhibitor overlying a typical pelvic radiotherapy plan.

response genes, including those coding for tumour necrosis factor ␣ (TNF␣), platelet-derived growth factor (PDGF), transforming growth factor ␤ (TGF␤), and basic fibroblast growth factor (bFGF). Many of these genes modulate radiosensitivity. Apoptosis, or programmed cell death, is the cell’s response to detection of DNA damage by radiation. This self-destruct mechanism stops cells perpetuating genetic mutations that might be harmful to the whole organism. In normal mammalian tissues some cells are susceptible to natural apoptosis, and the observation that these cells are generally radiosensitive implies a crucial role for apoptosis in determining radiosensitivity. The plasma-membrane-derived sphingomyelin pathway is one of the pathways that mediates radiation-induced apoptosis. Radiation activates the enzyme sphingomyelinase, which hydrolyses plasma-derived sphingomyelin and produces ceramide – a process that occurs within seconds of irradiation. Evidence for this mechanism came from the observation that synthetic ceramide analogues could mimic radiation-induced apoptosis in cellular systems (Figure 2).2 Conversely, cells from patients with Niemann-Pick disease (a severe neurological disorder caused by an inherited deficiency of sphingomyelinase) are resistant to radiation-induced apoptosis, but this resistance can be reversed by restoration of sphingomyelinase activity by retroviral transfer of human sphingomyelinase cDNA.3 How ceramide initiates apoptosis is not yet fully understood, but the process does involve a cascade of kinases and transcription factors; stress-activated protein kinase (SAPK) CRL is a Clinical Research Fellow at Charing Cross Hospital, London, UK. WJG is Professor of Cancer Biology at the University of Kent and Canterbury, Canterbury, UK. Correspondence: Professor William J Gullick, Department of Biosciences, University of Kent and Canterbury, Canterbury, Kent CT2 7NJ, UK. THE LANCET Oncology Vol 2 June 2001

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Review

Radiotherapy and cellular signalling

Ataxia telangiectasia The rare genetic disorder ataxia telangiectasia (AT) has interested radiation oncologists because people with this condition have hypersensitivity to ionising radiation (Figure 3). It manifests clinically with oculocutaneous telangiectasia and cerebellar ataxia, and patients show a high frequency of malignancy. At the cellular level, the AT phenotype is typified by defective G1/S and G2/M cell-cycle checkpoints. These defects allow damaged DNA, which would normally be repaired during a period of cell-cycle arrest, to replicate. ATM (the recently cloned gene mutated in AT) is a member of the phosphatidylinositol 3 kinase (PI3K) family that takes part in DNA damage surveillance and repair. Cell lines from AT patients show hypersensitivity to radiation-induced apoptosis, consistent with the concept that the ATM gene may function as an inhibitor of DNAdamage-induced apoptosis.5 In a recent study, B-cell lines from AT patients immortalised with Epstein-Barr virus showed increased radiation-induced activation of ceramide synthase, ceramide generation, and apoptosis.6 Cell lines from normal individuals did not show such responses. Transfection of the AT cell lines with wild-type ATM cDNA reversed the radiation hypersensitivity. Similarly, transfection of normal B cells with an antisense construct of ATM cDNA resulted in inactivation of the ATM gene, thereby conferring the AT phenotype on these cells. ATM does not appear to regulate sphingomyelinase-mediated apoptosis, which is initiated at the plasma membrane. Not all studies have attributed the AT phenotype to a modification of apoptosis and, although this mechanism THE LANCET Oncology Vol 2 June 2001

Plasma membrane sphingomyelin

X-irradiation

Sphingomyelinase

DNA damage

Sphinganine + FACoA

Ceramide

Ceramide synthase

Apoptosis

Figure 2. Radiation targets both DNA and plasma membrane to induce two different signalling systems for apoptosis via the same second messenger, ceramide. FACoA, fatty acid-coenzyme A.

may be important in some cell types such as lymphocytes, AT fibroblasts adopt a different approach.7

P53 tumour supressor gene The product of the P53 tumour suppressor gene is a 53 kDa nuclear phosphoprotein capable of inducing G1 cell-cycle growth arrest or apoptosis. It is stimulated by a variety of stress signals, including radiation, and eliminates damaged cells from the host. Mutations in the P53 gene are present in many human tumours and are associated with rapid tumour progression, and resistance to radiation therapy. Thus, it provides a potential target for intervention. Restoration of wild-type P53 should reverse resistance to apoptosis, but has proved difficult to achieve. Fibroblasts taken from normal mice showed no differences in radiosensitivity compared with those from P53 knockout transgenic mice with both

1.0 Fraction of cells that survive

is a critical part of this cascade. Radiation also activates apoptosis by a P53-dependent signalling system which is independent of the membrane-based sphingomyelin pathway and secondary to DNA damage. Ceramide is a second messenger in this pathway as well, but it is produced by activation of ceramide synthase rather than sphingomyelinase. Treatment of cells with the fungal toxin fumonisin B1 (a potent inhibitor of ceremide synthase) inhibits DNA-mediated apoptosis.4 There are several counterbalances to radiation-induced cell death. Although activation of the SAPK pathway leads to apoptosis, another cellular signalling pathway, the mitogenactivated protein kinase (MAPK) pathway, leads to cell survival and differentiation. Thus, the balance between the two pathways may dictate whether a cell survives or undergoes apoptosis, with the amount of damage required to shift the balance differing between cell lines. The MAPK pathway is activated by a number of growth factors and mitogens. Generally speaking, agonists that stimulate the MAPK pathway only weakly activate the SAPK cascade, with the reverse true for agonists of the SAPK pathway. If we understand how these complex cascades interact, we might be able to activate the apoptotic programme selectively in tumour cells but not in normal tissue, thus preventing damage to healthy cells. Various kinase and phosphatase inhibitors are emerging, as well as growth factors, cytokines and gene-based therapies, that can alter the apoptotic response of cells in vitro, and more recently, in vivo. A few of these are discussed in detail below.

Normal

0.1

Ataxia telangiectasia

0.01

0.001

0

4

8

12

16

Radiation dose (Gy) Figure 3. Cellular survival curves for a normal person and one with ataxia telangiectasia.

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Review

Radiotherapy and cellular signalling

C225 antibody TGF␣ EGFR

T

T RAS RAF1 MEK

Activated protein

MAPK

farnesylation by the enzyme farnesyl transferase. The advent of farnesyl transferase inhibitors, such as FTI-277, has opened up this cellular pathway to manipulation. FTI-277 sensitises cells with oncogenic mutations in their RAS genes to radiation, but has no effect on the survival of control cells.12,13 A similar effect is seen with inhibition of RAF1 protein kinase (another enzyme with a central role in promoting cellular mitogenesis) by an antisense oligodeoxyribonucleotide, resulting in sensitisation of cells to irradiation.14 In vivo studies with the antisense oligodeoxyribonucleotide in a liposomal formulation have confirmed significant tumour regression in response to irradiation of tumour-bearing athymic mice, well in excess of that seen with radiation alone.15

Growth factors Cytoplasm

Nucleus

Figure 4. Simplified diagram of the epidermal growth factor receptor (EGFR) with competing ligand TGF␣ and C225 antibody. Activation with ligand stimulates the MAPK signal transduction cascade to the nucleus, with resultant gene activation. T, Tyrosine kinase; MAPK, mitogen activated protein kinase; MEK, mitogen extracellular-regulated kinase.

P53 genes deleted.8 Likewise, transfection of mutant P53 into RKO colon carcinoma cells with normal P53 function did not alter radiosensitivity despite preventing radiationinduced G1 arrest. Other studies have shown some correlation between radioresistance and loss of G1 arrest linked to P53 mutation.9 These apparently contradictory results suggest that the effect of P53 on radiosensitivity may be cell-type specific. An alternative approach focusing on the prevention of apoptosis in normal tissues has, however, proved more successful. A treatment based on this principle may enable patients to receive effective therapy that would normally be limited by the damage incurred to normal tissues. One group has used a chemical inhibitor of P53 called pifithrin ␣ (an abbreviation derived from p-fifty-three inhibitor) for such a purpose. Mice pretreated with intraperitoneal pifithrin ␣ were compared to controls, with both groups receiving lethal and sublethal doses of whole-body ␥ irradiation. Pifithrin ␣ treatment completely rescued mice from lethal doses of radiation that normally kill 60% of animals, without promoting tumour formation.10 Obviously, patients whose tumours contain an active P53 gene would be ineligible for treatment with such a drug, since it might also protect their tumours from radiation therapy. Nevertheless, about half of all human cancers possess an inactive P53 gene and these patients would benefit from such intervention.

RAS oncogene The RAS oncogene has long been implicated in cellular radioresistance. Indeed, transfection of cells with activated RAS appears to increase radiation resistance.11 RAS proteins are processed in a series of reactions that require 368

Another area of potential therapeutic interest is the interplay between growth factors and radiation. The MAPK cascade can be activated by irradiation via a host of growth factors. It represents an adaptive cellular response to radiation that manifests itself as the accelerated cellular repopulation as seen with typical fractionated radiotherapy schemes. The epidermal growth factor receptor (EGFR) system represents a promising target since it is commonly overexpressed in many human tumours such as those of the breast, colon, lung, and head and neck.16 Cells with enhanced autocrine growth after irradiation show upregulation of both the EGFR and one of its principal binding ligands, TGF␣.17 EGFR tyrosine phosphorylation is stimulated by irradiation, and subsequent activation of MAPK shows that it mimics ligand binding.18 Human carcinoma cells that are not lethally damaged by irradiation can thereby give a proliferation response which can be opposed by specific inhibitors of EGFR autophosphorylation, such as the tyrphostin AG1478.19 Similarly, the use of C-225 (a monoclonal antibody that blocks ligand binding to the EGFR and thus inhibits activation of the receptor tyrosine kinase) enhances the radiosensitivity of certain cell lines and amplifies radiationinduced apoptosis (Figure 4).20 In a recent clinical study of 15 patients with locally advanced head and neck tumours, a combination of C-225 and radiation therapy was given.21 Thirteen (87%) patients experienced a complete response, well in excess of that expected for a similar population group (expected range 31–46%). The response also appeared durable; 67% of patients had progression-free disease at a medium duration of 13.9 months. C-225 was given as a loading dose 1 week before radiotherapy started and once weekly throughout the course of treatment. The combined regimen was well tolerated and represents a major advance in the transfer of laboratory-based research to the clinic. A phase III randomised clinical trial comparing C-225 and radiotherapy with radiotherapy alone in head and neck squamous cancers, is underway. However, data from studies with human squamous-cell carcinoma xenografts suggest that the effect of combining C-225 with radiation may derive from more than simply the antiproliferative and cellcycle effects of EGFR-system inhibition. In addition, C-225 may influence the capacity of these tumours to affect DNA repair after radiation exposure and inhibit expression THE LANCET Oncology Vol 2 June 2001

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Review

Radiotherapy and cellular signalling

of tumour angiogenesis markers such as vascular endothelial growth factor (VEGF).22 Part of another important growth regulatory system, the HER2 (also known as cERBb2) growth factor receptor is amplified and overexpressed in 25–30% of human breast cancers, and is associated with a poor clinical outcome.23 Monoclonal antibodies against the extracellular domain of HER2 inhibit the growth of breast cancer cells and enhance tumour sensitivity to radiation, a feature not seen in cells without HER2 overexpression.24 This selectivity is one of the potential benefits of such an approach. Many tumours also produce VEGF, a potent mitogen for vascular endothelial cells.25 Ionising radiation induces VEGF expression in mouse models; production of VEGF is thought to be a protective response of endothelium to irradiation. When tumour-bearing mice were injected with antibody to VEGF, irradiation appeared more effective in treating the tumours without affecting the intrinsic radiosensitivity of the tumour cells.26 The investigators concluded that radiation-induced VEGF production protects tumour vasculature from radiation, thereby increasing tumour radioresistance. Basic fibroblast growth factor (bFGF) has been used for preventing apoptosis in normal tissues following the observation that it protects endothelial cells from radiationinduced apoptosis in vitro.27 Mice injected intravenously with bFGF before and after whole-lung irradiation, were protected from the lethal radiation pneumonitis seen in irradiated control animals. bFGF binds with high affinity to vascular endothelium without leaking into perivascular spaces. This entrapment of bFGF in the vascular intima might be exploited to offer a differential radioprotective effect on normal vascular tissue as opposed to tumour tissue.28 Synthetic compounds can also be used to inhibit the MAPK pathway. MAPK inhibition with the synthetic compound PD-98059 in vitro trebled the number of DNA double-strand breaks resulting from 1 Gy of irradiation, confirming the MAPK cascade as a key cytoprotective pathway that is activated in response to commonly used radiation doses.29 So far we have concentrated on manipulation of the acute effects of ionising radiation, but it also produces a host of chronic late effects, one of which is tissue fibrosis. Tissue fibrosis is an occasional, but unfortunate, side-effect of both therapeutic exposure and accidental overexposure to radiation. It manifests in many organs such as the skin, heart, liver, kidney, and lung, with progressive loss of function over months or years, and is lethal in a substantial number of patients. It is characterised by a massive deposition of extracellular matrix and excessive fibroblast proliferation. Until recently, such fibrotic tissue was considered irreversible with little success from treatments such as corticosteroids. New data, however, challenge this idea. In particular, TGF␤ appears central to the development of tissue fibrosis; very high concentrations of TGF␤ can be detected hours after irradiation and persist for months or even years. The in vivo administration of TGF␤ to healthy animals not exposed to radiation produces tissue fibrosis,30 and conversely, liposomal Cu/Zn superoxide dismutase, an agent that downregulates TGF␤ secretion by myofibroblasts, reverses long-standing radiation-induced tissue fibrosis in human beings.31 THE LANCET Oncology Vol 2 June 2001

Radiation-induced gene therapy Finally, a more speculative, but nevertheless interesting, area involves the use of radiation-induced gene therapy. Briefly, the principle involves the use of a radiation-inducible promoter attached to a gene, the product of which might be able to express a protein toxic to the cell, or to activate a prodrug. This gene would have to reach a large number of target cells via a vector and remain in the cells for a sufficient period of time to be activated by radiation. Furthermore, it must by selective for tumour cells over normal cells to have any therapeutic potential. A phase I trial combining intravenous TNF␣ with therapeutic irradiation showed promising results in local tumour control and provided the basis for a investigation into this approach.32 However, in this trial, therapeutic efficacy was limited by the systemic toxicity of TNF␣. The rationale behind the gene-therapy approach was that high concentrations of TNF␣ can be induced inside the tumour by locoregional radiation exposure, while limiting systemic toxicity. The radiationinducible promoter region of the EGR1 gene was linked to the gene encoding TNF␣.33 An adenovirus vector was used to deliver the EGR1-TNF␣ construct to human tumours growing in nude mice. Subsequent irradiation caused large increases in intratumoral TNF␣ and increased tumour control compared with mice treated with radiation or EGR1-TNF␣ alone.33 The data confirmed that TNF␣ production was localised to the tumour bed resulting in no systemic toxicity and minimal local toxicity.

Conclusions Radiation oncologists often question whether molecular biology can offer any real benefit to their field. But technological advances in molecular oncology over the past decade are now poised to deliver tangible gains in the clinical setting. Compounds targeting signal transduction pathways have shown activity in clinical studies and their combination with radiotherapy holds promise. Such molecular targets might be inhibited or activated to manipulate the radiation response, for example, selectively inducing apoptosis in tumours or preventing it in adjacent normal tissue. Different cellular systems respond to radiation in different ways and thus one might expect disparate responses to these agents and approaches. However, despite this note of caution, the race to reproduce in the clinical setting the good preclinical data combining radiation and molecular therapeutics, is now underway.

Search strategy and selection criteria Data for this review were identified by searches of PubMed and MEDLINE from 1985 onwards. Only papers published in English were included. Search terms included: ‘radiation’, ‘cellular signalling’, ‘ataxia telangiectasia’, ‘RAS’, ‘P53’, and ‘growth factors’. In addition, the proceedings of the annual meetings of the American Association of Cancer Research and the American Society for Clinical Oncology from 1995–2000, were searched.

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Review References

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