Molecular Cancer and Radiation Biology - Perez & Brady's Principles and Practice of Radiation Oncology (Perez and Bradys Principles and Practice of Radiation Oncology), 6 Ed.

Perez & Brady's Principles and Practice of Radiation Oncology (Perez and Bradys Principles and Practice of Radiation Oncology), 6 Ed.

Chapter 3. Molecular Cancer and Radiation Biology

Michael Baumann, Nils Cordes, Mechthild Krause, and Daniel Zips

During the last three decades molecular cancer research has become a rapidly growing branch of the biomedical sciences. Many advances in our understanding of cancer are closely related to innovations in biotechnology—for example, techniques to knock out or to knock in a specific gene of interest, experimental maneuvers to manipulate temporal and spatial gene expression, the development of high-throughput methods to study the entire genome and proteome of cancer cells, and a vastly growing methodology to specifically interfere with signal transduction. This chapter provides an introductory overview of molecular cancer and radiation biology.

Cell biology approaches contribute to modern radiobiology and help to better understand effects of ionizing radiation on cells, tumors, and normal tissues. Knowledge in molecular cancer biology is important for clinical decision making in oncology and the development of novel biology-driven strategies in the multidisciplinary clinical environment.¹^,² Molecular pathology of tumors increasingly supplements classic histopathology and immunohistochemistry, thereby providing the basis for improved treatment stratification in oncology.³^,⁴ Molecular pathophysiology describes mechanisms leading to a characteristic microenvironment of tumors or the mechanisms that eventually lead to the manifestation of radiation sequelae in normal tissues.⁵ Molecular imaging has become important not only for staging but also for biologic characterization of tumors and for determination of target volumes in radiation oncology, including new approaches such as dose painting.⁶^–⁸ Molecular targeting in radiotherapy may either increase the tumor response or protect normal tissues, thereby enhancing the therapeutic gain of the treatment.⁹^–¹⁰^,¹¹

Compared to other fields of oncology, radiotherapy appears to be particularly promising to integrate molecular targeting approaches.¹¹ First, the radiobiologic mechanisms of the response of tumors and normal tissues to radiotherapy are well characterized, and the molecular pathways involved in these responses are increasingly known. Second, similar to conventional chemotherapeutic drugs, the novel drugs developed so far are not curative in themselves. In contrast, radiotherapy in itself is extremely efficient in eradicating cancer stem cells, and recurrences often occur from only one or a few surviving cancer stem cells.^12,¹³^–¹⁴ Thus, even if novel drugs have only the potential to kill a limited number of cancer stem cells, this might be sufficient to increase local control when combined with radiotherapy. The same argument applies when these drugs increase the radiosensitivity of tumor cells or when normal tissues are specifically protected. Third, in contrast to systemic chemotherapy, radiotherapy can be modulated in dose, time, and space. This allows individual tailoring of the effects of combined treatments in consideration of the spatial distribution of cancer stem cell burden as well as with consideration of normal tissues. Preclinical data and early clinical results corroborate these arguments¹⁵^–¹⁹ and support further translational research on biologically enhanced radiotherapy.

PRINCIPLES OF MOLECULAR CANCER BIOLOGY

The majority of human cancers arise from single somatic cells as a result of a stepwise evolutionary process of accumulation of multiple genetic and epigenetic aberrations.²⁰ Genetic changes include point mutation, deletion, insertion, gene amplification, chromosomal instability, loss of heterozygosity, and translocation. Silencing of tumor suppressor genes by promoter hypermethylation, histone modifications, and microRNA (noncoding RNA) expression represents important epigenetic mechanisms of tumorigenesis.²¹^,²² Infection with oncogenic viruses might contribute to the development of human cancer, for example, human papilloma viruses in cervical or head and neck cancer, hepatitis B viruses in hepatocellular carcinoma, and human immunodeficiency viruses in Kaposi’s sarcoma.²³^,²⁴ The hostile micromilieu in solid tumors, particularly hypoxia, further promotes progressive genomic alterations and clonal selection.²⁵ Consequently, cells gain advantage in proliferation and survival, which eventually results in malignant transformation, a prerequisite for the development of cancer and metastatic spread.

Conceptually, two classes of cancer genes can be distinguished. First, oncogenes are activated in cancer cells by genetic alterations resulting in a gain of function. Mutations in proto-oncogenes acting in a dominant fashion (i.e., a genetic alteration in one of the alleles) are sufficient for gene activation. Typical oncogene functions are stimulation of cell proliferation (e.g., by activation of Ras) and increase in cell survival (e.g., by activation of PI3K/Akt signaling). Disorders in the second class of cancer genes, tumor suppressor genes, cause a loss of function. Mutations in one allele of a tumor suppressor gene are recessive because they can be functionally compensated by the second, nonmutated (wild-type) allele. While some cancers can be attributed to single genetic alterations, most sporadic solid tumors exhibit a wide range of disorders in numerous cancer genes. Simplified mathematical modeling of the increasing incidence of common cancers as a function of age suggests that four to seven somatic gene alterations are required for carcinogenesis.²⁰^,²⁶ A typical example for multistep tumorigenesis is the adenoma–carcinoma sequence in colorectal cancer.²⁷

Inherited cancer predisposition can be divided into the rare group of inherited cancer syndromes and familial cancers (strong predisposition) and the more frequent group of predisposition without evident family clustering (weak predisposition).²⁸ The first group includes syndromes caused by germline mutations affecting DNA repair, genomic stability, and cell cycle control, for example, TP53 (Li–Fraumeni syndrome), nucleotide excision repair genes (xeroderma pigmentosum), ATM (ataxia telangiectasia), DNA mismatch repair genes (hereditary nonpolyposis colorectal cancer), and BRCA 1/2 (familial breast cancer). An example of a familial cancer syndrome related to oncogene activation is neurofibromatosis type I, in which the mutated NF1 gene results in activation of the Ras oncogene.²⁸ Germline mutations causing inactivation of the cell–cell adhesion molecule E-cadherin can be found, for example, in familial diffuse gastric carcinoma.²⁹

The essential molecular biology of cancer can be summarized by a simplified concept based on a small number of underlying principles. In their seminal articles, Hanahan and Weinberg³⁰^,³¹ described these principles shared by most human tumor types as the “six hallmarks of cancer” (Table 3.1). These six hallmarks are the consequence of specific genetic alterations in important oncogenes and tumor suppressor genes.

The Hallmarks of Cancer

Cancer is characterized by the loss of growth control due to acquired capabilities of autonomy of growth signaling, deteriorations in the cell cycle regulation, insensitivity to growth-inhibitory signals, evasion of apoptotic cell death, induction of neoangiogenesis, and activation of invasive growth and metastasis.³⁰^,³¹ Normal cell division is tightly regulated by stimulatory and inhibitory growth signals. Growth signaling involves interaction of diffusible growth factors or cytokines with transmembrane receptors, as well as regulation of growth by components of the extracellular matrix and by cell–cell interactions. Once a resting cell receives a sufficient growth stimulus, it enters the cell cycle by passing the restriction point and four distinct phases of cytokinesis and mitogenesis: gap-1 (G1), DNA synthesis (S), gap-2 (G2), and mitosis (M).³² Passage through the restriction point and entry into S and M (G1/S and G2/M checkpoints) are governed by several proto-oncogenes and tumor suppressor genes. Each phase of the cell cycle is regulated by specific complexes of cyclins (cyclins A to E) and their respective partners, the cyclin-dependent kinases (cdks). Important complexes are cyclin D/cdk4 and cyclin D/cdk6 (restriction point, G1), cyclin E/cdk2 (G1/S), cyclin A/cdk2 (S, G2), and cyclinB/cdk1 (M). There is increasing evidence that many functions of the cyclins are compensatorily covered among the cyclin family, that is, in the absence of one cyclin, other cyclins are able to act in different cell cycle phases.³³^,³⁴ Cyclins are directly upregulated by growth factors and indirectly via c-myc and Ras. Several cyclin/cdk complexes phosphorylate the retinoblastoma protein (pRB), facilitating the G1/S transition, as well as blocking differentiation. The activity of the cdks is regulated by phosphorylation and dephosphorylation events, as well as by interaction with various inhibitory molecules belonging to the INK4 (p15, p16, p18, and p19) and Cip1/Kip1 (p21, p27, and p57) families. The proteins p15 and p21 are related to two important tumor suppressors, the antiproliferative factor transforming growth factor-β (TGF-β) and p53, respectively.

TABLE 3.1 THE SIX HALLMARKS OF CANCER WITH INVOLVED ONCOGENIC AND TUMOR-SUPPRESSIVE MOLECULES (SELECTION)

Self-Sufficiency in Growth Signals

Mitogenic signals from growth factors, cytokines, extracellular matrix, and cell–cell adhesion molecules are transferred into the cell by different classes of transmembrane receptors.³⁰^,³¹ Malignant cells acquire the capability to escape from the tightly regulated dependence on extracellular growth signals. The molecular mechanisms include overexpression of growth factors as well as growth factor receptors (autocrine and paracrine stimulation), receptor mutations leading to constitutive receptor activation without ligand binding, and molecular aberrations in the intracellular signal transduction pathways.

Receptor tyrosine kinases (RTKs) represent a group of oncogenic, transmembrane receptors consisting of an extracellular ligand-binding domain, a transmembrane part, and an intracellular catalytic domain with tyrosine kinase activity.³⁵ On ligand binding and receptor homo/heterodimerization, the protein kinase is subsequently activated, resulting in phosphorylation of tyrosine residues of the receptor itself (autophosphorylation) or target proteins. Depending on the cellular context, this triggers an intracellular signaling cascade that eventually leads to proliferation, survival, differentiation, and migration. Among the known human RTKs are the epidermal growth factor receptors (EGFRs), the platelet-derived growth factor receptors (PDGFRs), the vascular endothelial growth factor receptors (VEGFRs), the fibroblast growth factor receptors, the ephrin receptors, and tyrosine kinase receptor. The EGFR family consists of four distinct members (EGFR/ErbB-1, HER2/ErbB-2, HER3/ErbB-3, and HER4/ErbB-4).³⁶ The receptor ligands, such as epidermal growth factor, tumor growth factor-α, and neuroregulin-1, as well as their cognate receptors, are abundantly expressed in a large variety of human cancers, including lung, breast, head and neck, and gliomas, and have been related to poor prognosis. This finding led to the recognition of the EGFRs as important targets for cancer therapy. Mechanisms underlying the increased activation of the EGFR pathway in cancer cells include gene amplification and activating mutations, for example, EGFRvIII, a constitutively ligand-independent EGFR mutant.³⁶

RTK-Initiated Signal Transduction

The extracellular signals received by the RTKs are translated into a large variety of different cellular responses by a cascade of molecular processes, that is, signal transduction.³⁵ This RTK signaling involves several distinct molecular pathways that are often deregulated in cancer cells. Prime examples of such deregulated pathways are the Ras/RAF/mitogen-activated protein kinase (MAPK) pathway, the phosphoinositide 3′ kinase (PI3K)/Akt pathway, the jak/stat molecules, and protein kinase C. Cooperative and mutual cross-talk between the different transduction pathways forms a complex signaling network.

An important signal transduction route that has been extensively studied in EGFR signaling represents the Ras/Raf/MAPK pathway. The Ras proteins (H-Ras, K-Ras4A, K-Ras4B, and N-Ras) are GTPases and are attached in their active state (GTP bound) to the inner surface of the cell membrane. The Ras proto-oncogene, predominantly K-Ras, is mutated in about one-third of human cancers. Activating mutations are frequent in adenocarcinomas of the pancreas (up to 90%), colorectum (about 50%), and the lung (about 30%).³⁷ The processing and membrane attachment of the functional Ras are governed by farnesyltransferases, which are the molecular target for specific pharmaceutical inhibitors of activated Ras.³⁸ EGFR activation channels via the Grb2/SOS complex to the Ras molecule. Activated Ras binds to Raf, facilitating its function as a serine/threonine kinase. Constitutively activated Raf proteins as a result of genetic alterations in the Raf genes have been shown in a large variety of human malignancies.³⁹ Raf associates with MEK1/2 kinases, which in turn activate MAPKs. Activated MAPKs are subsequently translocated to the nucleus and initiate transcription by the activation of several transcription factors, for example, Elk-1. Consequently, gene expression is changed, and proliferative processes are triggered.

In addition to Raf, activated Ras has an effect on various downstream molecules, including PI3K.⁴⁰ Following either direct activation by RTKs or indirect Raf-mediated activation, this group of kinases phosphorylates the 3′-OH group of the inositol ring in inositol phospholipids. Signaling mediator molecules of PI3K include phosphoinositide-dependent kinase 1, Akt, protein kinase C, and subsequently the nuclear transcription factor NFκB. The cellular responses on activation of the PI3K pathway are cell-type specific and include changes in gene expression, cell cycle progression, survival, and apoptosis. The last has been linked to PKB/Akt negatively controlling apoptosis-regulating molecules, such as Bad, caspase 9, Fas ligand, cAMP responsive element–binding protein, and IκB kinase. The tumor suppressor protein PTEN negatively regulates the PI3K/Akt pathway. Akt overexpression or hyperactivity and PTEN mutations are found in numerous human malignancies.⁴¹ Akt phosphorylates, the molecular target of rapamycin, a compound that exhibits anticancer activity.⁴²

Cytokine Receptors

A large number of growth-stimulating hormones, growth factors, and cytokines such as the interleukins, erythropoietin, and prolactin bind to the class of cytokine receptors that are structurally different from the previously described RTKs.⁴³ The intracellular signaling in response to cytokine receptor activation includes the JAK and STAT kinases. In addition to the cytokine receptors, many RTKs can activate the JAK/STAT pathway. In malignant cells the autonomy of growth signaling can be associated with mutations in cytokine receptors (e.g., in the erythropoietin receptor) and constitutively upregulated activity of the JAK/STAT pathway in hematopoietic malignancies. STAT activation may result from transformation of tyrosine kinases such as v-src, v-Abl, and Bcr/Abl by viral oncoproteins from human T-cell lymphotropic virus or Epstein–Barr virus.⁴⁴

Wnt Signaling

Wnt proteins are diffusible growth factors that bind to specific surface receptors (Frizzeled) and trigger distinct intracellular pathways leading to cell growth.⁴⁵ Wnt signaling involves the tumor suppressor protein adenomatous polyposis coli (APC) and regulates by phosphorylation the steady-state levels of cytosolic β-catenin. An increased level of β-catenin facilitates its transit into the nucleus, activation of transcription factors, and subsequently transcription of β-catenin target genes, including c-myc, c-jun, and cyclin D1. The aberrant activation of Wnt signaling caused by mutations in the β-catenin and APC genes are typical findings in colon cancer and melanoma but also have been identified in a large variety of other human cancers. Wnt signaling has been shown to play an important role in tumorigenesis and in cancer stem cell self-renewal and differentiation and thus provides a promising target for anticancer treatment.⁴⁶

Cytoplasmic, nonreceptor kinases are often activated in malignant disease. For example, the Bcr/Abl fusion protein kinase resulting from the translocation t(9;22) is a typical molecular finding in chronic myeloid leukemia and represents the molecular target for small-molecule inhibitors such as imatinib.⁴⁷ This drug also inhibits c-KIT, a cytoplasmic tyrosine kinase that has been shown to be activated by mutation in gastrointestinal stroma tumors, thus providing another molecular targeting approach.⁴⁸

Transcription factors bind to the DNA and activate the expression of specific genes. Oncogenic transcription factors are overactive in most human cancers and contribute to the autonomy of growth signaling. Based on their mechanism of activation, three groups can be separated.⁴⁹ First are the steroid receptors, which are found in hormone-sensitive breast or prostate cancer. The second group of transcription factors resides in the nucleus, is governed by kinase signals, and consists of various members, including myc, activator protein 1 (AP-1), and E2F. Myc transcription factors are induced in cancer by different mechanisms, including translocation and gene amplification. For example, c-myc is activated by the chromosomal translocation t (8;14), which brings the c-myc gene under the control of the immunoglobulin G enhancer, resulting in a constitutive c-myc expression in 80% of Burkitt lymphomas. N-myc and L-myc amplifications are found in neuroblastoma and small cell lung cancer, respectively. AP-1, consisting of JUN, FOS, ATF, and MAF, can exhibit oncogenic or antioncogenic effects depending on the cellular context.⁵⁰ The third group of oncogenic transcription factors—the latent transcription factors—is activated by ligand–receptor interactions and includes the STATs (see previous discussion), the WNT–β-catenin pathway (see previous discussion), NFκB, and the molecules Notch and Hedgehog. NFκB levels have been shown to be constitutively active in lymphomas, leukemias, and breast and colon cancer.⁴⁹

Loss of Cell Cycle Control

Retinoblastoma Tumor Suppressor Protein (pRB)

The retinoblastoma protein is an important regulator of cell cycling and cell differentiation.⁵¹ Loss of pRB function is often found in malignant cells and leads to an uncontrolled G1/S transition, genomic instability, and loss of differentiation. Hypophosphorylated pRB binds to several proteins, including the E2F transcription factors and histone deacetylases. These proteins are released on phosphorylation of pRB, for example, by cyclin D/cdk4, cyclin E/cdk2, and cyclin A/cdk2 complexes, and contribute to cell cycle transition and/or to inhibition of terminal differentiation. As a result, the cell is switched from a quiescent phenotype toward a proliferative state. Released E2F activates transcription of genes required for DNA synthesis such as dihydrofolate reductase, thymidylate reductase, and DNA polymerase. Loss of pRB function can result from mutation or inactivation by oncogenic viral proteins such as human papillomavirus (HPV) E7. Somatic mutations in the RB gene have been detected in a variety of epithelial and mesenchymal malignancies.⁵¹ Loss of cell cycle control by impairment of the pRB pathway can also result from overexpression of cyclin D, activating mutations and amplification of cdk4, and loss of p16 (INK4), leading to a constitutive expression of E2F target genes.⁵¹ Loss of function in the pRB pathway contributes to genomic instability by E2F-mediated overexpression of MAD2, an important component of the mitotic checkpoint.⁵² The encoding RB gene was the first characterized tumor suppressor gene involved in retinoblastoma, a rare childhood tumor. Retinoblastoma is a typical example for the two-hit model of tumor induction.²⁶ The first hit, that is, mutation in one RB allele, is either a germline (inherited form, often bilateral) or an acquired somatic mutation (sporadic form, mostly unilateral). An acquired somatic mutation in the other RB allele causes the inactivation of the pRB and consequently tumorigenesis.

TP53 tumor suppressor gene is inactivated in the majority of cancers and has been associated with poor prognosis in some cancers.⁵³ Wild-type p53 binds to specific DNA sequences and activates transcription of numerous genes, including MDM2, GADD45, p21^CIP–1, cyclin D, and the proapoptotic BAX. On the other hand, p53 can negatively regulate gene transcription (e.g., for genes such as myc, cyclin A, MDR1,and the antiapoptotic Bcl-2).⁵⁴ The role of p53 for the radiation response is discussed later in this chapter. Loss of p53 function has numerous biologic consequences, including loss of cell cycle arrest at the G1/S-phase checkpoint after genotoxic stress, inhibition of apoptosis, and differentiation.⁵⁴ Furthermore, loss of p53 function seems to be critical for chromosomal stability, as p53 is involved in DNA repair, recombination, and replication. Inactivation of the TP53 gene is the most common genetic alteration in human cancer. More than 80% of the p53 alterations result from missense mutations in the DNA-binding domain.⁵⁵ The obvious predominance of specific missense mutations in human tumors with the presence of a full-length protein suggests an additional role of mutant p53 as an oncogenic protein with gain-of-function and dominant-negative properties. In addition to mutations, p53 function can be compromised by aberration of its negative regulator MDM2 or by viral proteins HPV E6/E7.⁵⁶ While most of the TP53mutations are sporadic, a high proportion of patients with Li–Fraumeni syndrome, a rare cancer syndrome with a spectrum of carcinomas and sarcomas at early age, carry germline mutations.

Insensitivity to Growth-Inhibitory Signals

TGF-β regulates multiple cell functions, such as proliferation, extracellular matrix synthesis, angiogenesis, immune response, apoptosis, and differentiation.⁵⁷ The TGF-β family (TGF-β1 to 5) belongs to the superfamily of peptide hormones. The different TGF-β isoforms bind to specific cell surface receptors (TβRI to TβRV). TGF-β–mediated growth inhibition is associated with activation and repression of target gene transcription. Genes of proproliferative kinases such as cdks are repressed, whereas genes of the major cyclin-dependent kinase inhibitors p15 and p21 are transcriptionally activated. Ligand binding to TβRV stimulates serine/threonine-specific phosphatases, which inhibit proliferation by dephosphorylation of pRB. Alteration of TGF-β signaling in tumors, for example, by mutations and transcriptional silencing leads to insensitivity to inhibitory ligands.

TABLE 3.2 TYPES OF RADIATION-INDUCED CELL DEATH AND THEIR CHARACTERISTICS

Resistance to Apoptosis

Under physiologic conditions, tissue homeostasis results from the balance of cell division and cell loss. This balance is disturbed in most tumors by an increased cell division rate due to the molecular mechanisms previously described and by an inappropriate rate of cell death, such as by senescence and apoptosis⁵³^,⁵⁸ (Table 3.2). Apoptosis is a complex, multistep process involving numerous molecules, including adapter proteins, members of the Bcl-2family, and cysteine-aspartate proteases (caspases).⁵⁹ The last are divided into initiator (apical) caspases (caspases 1, 2, 4, 5, 8 to 10, 12) and effector (executioner) caspases (caspases 3, 6, 7, 11, 13). Caspases activate specific substrates by proteolytic cleavage. The activity of caspases is regulated by heat-shock proteins and inhibitor of apoptosis proteins. The executive phase of the apoptotic program includes the release of cytochrome C from mitochondria after membrane depolarization, formation of the apoptosome complex (Apaf-1, pro–caspase 9, cytochrome C), and activation of effector caspases, leading subsequently to morphologic changes (e.g., DNA condensation and fragmentation). Initiator caspases are activated by two major pathways—the extrinsic, receptor-mediated pathway and the intrinsic, mitochondria-mediated pathway. The extrinsic apoptotic signaling starts from binding of death ligands, such as Fas, tumor necrosis factor (TNF), and TRAIL, to their corresponding cell-surface receptors. Subsequently, a cell type–specific intracellular program is executed, including caspases, and members of the Bcl-2 family (Bax, Bid, Bak) eventually trigger the release of cytochrome C (type II) or directly activate effector caspase 3 (type I). The intrinsic, mitochondria-mediated pathway is triggered in response to cellular stress, such as DNA damage, chemotherapy, ionizing radiation, growth factor withdrawal, and kinase inhibition. While the acquired capability to escape from apoptosis is a prerequisite for tumorigenesis, it does not necessarily correlate with resistance to cancer treatment.⁵³

An important mediator between the recognition of DNA alterations and apoptosis is the tumor suppressor p53.⁵⁴ On DNA damage, p53 is stabilized and induces cell cycle arrest (see previous discussion), senescence, or apoptosis. Promotion of apoptosis by p53 results from transcriptional repression of antiapoptotic proteins (e.g., Bcl-2 and survivin) and activation of the proapoptotic proteins, including Bax, PUMA, and NOXA. Of importance, the apoptotic pathways are closely linked to protein kinase signaling. Thus, protein kinases such as MAPK and PI3K/Akt (see previous discussion) are able to modulate the balance between death and survival stimuli in the cell in a context-dependent manner. Given the complex regulation of apoptosis, it is not surprising that a large variety of molecular alterations can result in an escape of cancer cells from this type of cell death.⁶⁰ For instance, the extrinsic pathway is affected by downregulation of caspase 8 activity by promoter methylation, mutation, and upregulation of the negative regulator FLIP. Many tumor cells show a deregulated intrinsic, mitochondria-related apoptotic pathway caused by an imbalance of expression levels of proaptoptotic and antiapoptotic proteins of the Bcl-2 family. Abnormal expression levels of inhibitor of apoptosis proteins (Apaf-1) and heat-shock proteins result in an impaired execution phase of apoptosis in cancer cells. Moreover, important mediators and regulators of the apoptotic program, such as p53, PI3K/Akt, PTEN, and MAPK are often functionally deregulated. While it is well established that the escape from apoptosis represents an important and possibly essential step in tumorigenesis, the importance of this phenomenon for therapy resistance and outcome prediction and as a potential target to improve conventional cancer therapies remains unclear.⁵³^,⁶⁰

Limitless Replicative Potential

Loss of growth control and resistance to cell death are not sufficient for the development of macroscopic tumors.³¹ Normal cells have the capacity for a finite number of cell divisions, that is, a limited replicative potential. Thereafter cells either stop proliferating and enter the process of senescence, that is, a viable but nonproliferative state, or they die. During tumorigenesis, some premalignant cells become immortal by circumventing senescence, that is, by acquiring a capability of limitless replicative potential.³¹ Of importance, malignant tumors consist of heterogeneous populations differing in their replicative potential. While most cancer cells have a limited replicative potential, a small subpopulation (i.e., cancer stem cells) have an infinite replicative capacity to reconstitute the tumor.⁶¹ Thus, this subpopulation represents the target for curative cancer therapy, that is, all of these stem cells have to be inactivated by therapy to achieve cure. It remains to be clarified whether cancer stem cells represent a distinct tumor cell subpopulation or whether all cancer cells have a stem cell potential and can switch between different stages in response to stress or environmental conditions.

The molecular mechanisms underlying the escape from senescence and other modes of cell death include loss of tumor suppressor proteins such as p53 and pRB as well as telomere maintenance.³¹ Telomeres are chromatin segments located at the ends of the chromosomes that protect these regions from recombination and degradation.⁶² As telomeres are incompletely replicated, they become progressively shorter during each cell division, subsequently resulting in loss of chromosomal protection, which in turn triggers senescence. Cancer cells generally have shorter telomeres than normal cells but are able to perpetuate their replicative potential by expressing telomerase, a complex including DNA polymerase, which reconstitutes the telomeres.⁶²

Induction and Sustaining Angiogenesis

To grow beyond microscopically sized cell aggregates of 1 to 2 mm, tumors depend on angiogenesis. Proliferating cells require appropriate oxygen and nutrient supply, which is physiologically limited to a distance of 100 to 200 μm from the next blood vessel. Therefore, malignant cells must induce and sustain their own vascular system to form tumors and metastases.⁶³ The process of the angiogenic switchduring tumorigenesis (i.e., the transition from the avascular phase to the vascular stage) is governed by different molecular changes in tumor cells and in cells of the surrounding stroma. The major event of the angiogenic switch is that proangiogenic factors—mostly growth factors such as VEGFs, fibroblast growth factors, and PDGFs—outbalance antiangiogenic factors, such as thrombospondin (TSP-1), angiostatin, and endostatin. The driving forces toward the imbalance of angiogenic factors in tumors include activation of oncogenes; for example, Ras and myc activation results in VEGF upregulation and TSP-1 repression.⁶⁴^,⁶⁵ The loss of function in tumor suppressor proteins also contributes to the angiogenic switch by transcriptional regulation of angiogenic factors. For example, p53 upregulates TSP-1 and downregulates VEGF gene expression.³⁰ Hypoxia promotes tumor angiogenesis via the hypoxia inducible factor 1, which transcriptionally regulates many angiogenic molecules.⁶⁶^,⁶⁷ As originally proposed by Folkman,⁶⁸ effective targeting of molecules and pathways involved in angiogenesis has been shown to exhibit anticancer activity.⁶⁹

Tissue Invasion and Metastasis

Locoregional and distant spread of tumor cells requires detachment from the primary tumor, invasion into surrounding tissues, intravasation into blood or lymphatic vessels, adhesion to endothelial cells at distant sites, extravasation, and eventually colonization in distant organs. The acquired capability of cancer cells to grow invasively and to metastasize is associated with multiple genetic and biochemical alterations of the cell–cell and cell–matrix interactions. Tissue invasion and metastasis require, at different steps, contrary capabilities—for example, detachment from the primary tumor versus adhesion at the metastatic site—suggesting that rapid adaptations, genetic instability, and clonal selection play an important role. Gene expression profiling revealed metastatic signatures in primary tumors but also host polymorphisms, resulting in a genetically determined individual disposition to develop metastases.⁷⁰^,⁷¹ The often-observed nonrandom pattern of metastasis in different types of cancer led to the “seed and soil” hypothesis describing the complex interplay between cancer cells and environmental factors.⁷² Experimental data suggest that bone marrow–derived endothelial progenitor cells exhibit preconditioning functions for metastases; that is, they reside in tumor-specific metastatic sites before the colonization with tumor cells and prepare the optimal environment for tumor cell homing.⁷³

Many physiologic functions of the cell, such as proliferation, migration, survival, and differentiation, are regulated by interactions with neighboring cells and with molecules of the extracellular matrix (ECM). The ECM consists of a large variety of different components, including collagens, fibronectins, laminins, tenascin, proteoglycans, matrix proteases, and their specific inhibitors. Altered composition of the ECM is a typical finding in malignant tissues. For example, tenascin overexpression was found in the stroma of breast cancer, colon carcinoma, and glioma.⁷⁴ It has been suggested that, due to the antiadhesive properties of tenascin, this change in the ECM facilitates detachment of cancer cells and subsequently tissue invasion and metastasis. Matrix proteases are important for ECM turnover and enable tumor cells to degrade extracellular barriers, a prerequisite for tissue invasion and metastasis.⁷⁵ The different classes of matrix proteases include serine proteases (plasminogen activator, plasmin, and elastase), cysteine proteases (cathepsins), and matrix metalloproteinases (collagenases, stromelysins). The last are released from the cells in an inactive form and activated by binding to zinc ions. The activity of the matrix proteases, and thereby the ECM turnover rate, is also determined by inhibitors of the proteases such as tissue inhibitors of metalloproteases or plasminogen activator inhibitor. It has been shown that tissue invasion and metastasis in various types of cancer are associated with increased expression of matrix proteases and decreased activity of their inhibitors compared with normal cells.⁷⁶

Cell–cell and cell–matrix interactions are mediated by different adhesion molecules, including integrins, cadherins, immunoglobulin-like cell adhesion molecules, and the hyaluronan receptor CD44.⁷⁷ Not only do these molecules exert structural functions, but they also are essential for signal transduction. Integrins represent a class of heterodimeric transmembrane ECM receptors.⁷⁸ At least 24 integrin receptors are formed by 18 α and 8 β subunits with overlapping binding affinity to ECM components and functions. Further diversity in the integrins results from posttranslational modification such as alternative splicing. Intracellular signal transduction on binding of integrins to ECM components includes activation of the focal adhesion kinase and subsequent association with PI3K, which is required for focal adhesion kinase-promoted cell migration and survival. Other signaling partners of integrins include ILK, protein kinase C, Rho family of small G proteins, and cytoplasmic kinases (src, Abl), which contribute to activation of MAPK/JNK and subsequently stimulate proliferation and migration.

Besides the ability to bind ECM proteins such as fibronectin, collagen, or laminin, some integrins recognize members of the disintegrin and metalloproteinase (ADAM) family or counter receptors on neighboring cells such as immunoglobulin-type receptors like intercellular cell adhesion molecules or vascular cell adhesion molecules.⁷⁹ Cross-talk between integrin signaling, growth factor signaling, and tumor suppressor proteins such as PTEN and p53 have been described. The capability of tissue invasion and metastatic spread has been associated with altered integrin function in tumors.⁸⁰ In general, cancer cells show a more pronounced variability in integrin combinations, an abnormal expression pattern, and aberrant spatial expression compared with nonmalignant cells, facilitating interaction with ECM of different composition and thereby permitting cancer cell survival and proliferation at distant sites. Alternative splicing of the hyaluronan receptor CD44 results in a complex, cell type–specific expression pattern that has been demonstrated to be altered in many tumors and metastasis.⁸¹ E-Cadherin is a glycoprotein expressed at the cell surface and intracellularly linked to the cytoskeleton via catenin proteins. In addition, E-cadherin is connected to multiple intracellular signaling pathways. The function of E-cadherin as a tumor suppressor was established from experimental and clinical observations that a loss of function (e.g., by mutation, promoter methylation, increased proteolytic degradation by matrix metalloproteinases, increased endocytotic degradation induced by phosphorylation) is a frequent finding in human cancers and is associated with an invasive and metastatic phenotype.⁸² The multifunctional immunoglobulin-like cell adhesion molecules are expressed in a large variety of cell types. Some members of this superfamily, such as NCAM, CEA, Mel-CAM, and L1, are involved in tumorigenesis, metastasis, and invasion.⁷⁷

MOLECULAR RADIATION BIOLOGY

Target Molecules of Radiation Damage

Radiation effects may occur as direct ionizations in an organic molecule or indirectly via free radical processes. As cells consist mostly of water, most ionizations produced by irradiation occur in water molecules. Within only 10⁻¹⁰seconds, radiolysis of water leads, among other entities, to e⁻aq, H·, and OH·. About 60% to 70% of cellular DNA damage produced by ionizing radiation is caused by OH·.⁸³ The radiation-induced reactive oxygen species (ROS) undergo further reactions—for example, production of H₂O₂ from two hydroxyl radicals. Aerobes have evolved antioxidant defenses to protect themselves against the oxygen-derived species generated in vivo or from external sources. These defenses include enzymes (such as superoxide dismutases, catalase, and glutathione peroxidase), low–molecular mass agents (such as α-tocopherol and ascorbic acid), and proteins that bind metal ions in forms unable to catalyze the generation of free radicals. In contrast to those defensive mechanisms, the oxygen molecule has a high affinity to free radicals, which may give rise to further cascades of radical production and thereby to the fixation of free radical damage to important macromolecules of the cell (e.g., DNA). This is one explanation of the oxygen effect of radiation damage, that is, the fact that well-oxygenated cells are more radiosensitive than hypoxic cells.⁸⁴^,⁸⁵

By far the most important target for the biologic effects of ionizing radiation is the DNA (Fig. 3.1). This is obvious for the induction of mutations but has also been consistently demonstrated for the killing of cells in a number of different experiments. Irradiation of the cytoplasm of cells with short-range α-particles only leads to cell kill at very high doses, whereas 1,000-fold lower doses to the nucleus are sufficient to kill the cell.⁸⁶ Radioactive isotopes with short-range emission effectively kill cells when incorporated into the DNA but not when predominantly incorporated in cell membranes.⁸⁷ Modification of radiation-induced cell kill by different measures, including hypoxia, high–linear energy transfer radiation, or hyperthermia is linked closely with a change in the induction and repair of DNA double-strand breaks. Exposure of cells to about 1 Gy causes approximately 3,500 DNA injuries, 1,500 to 2,500 of which are damaged bases, 1,000 single-strand breaks (SSBs), 40 double-strand breaks (DSBs), and an estimated 100 to 200 local multiple-damaged sites, where one or several DSBs occur in close proximity to SSBs and base damage.⁸⁸ It will be shown later that most radiation-induced DNA damage is recognized and very efficiently repaired by the cell.

Besides effects on DNA, ionizing radiation also evokes biologically important responses on proteins (e.g., transmembrane receptors) and on lipids (e.g., ceramides) (Fig. 3.1). Radiation-induced activation of receptors will be discussed later. Ionizing irradiation induces rapid sphingomyelin hydrolysis by acid sphingomyelinase to generate ceramide, an inductor of apoptosis.⁸⁹

FIGURE 3.1. Simplified illustration of biologic effects of ionizing radiation mediated via DNA and non-DNA target molecules. ROS, reactive oxygen species.

FIGURE 3.2. Functional effects of ionizing radiation on cells. The categories are not exclusive.

Biologic Consequences of Irradiation

The effects of ionizing radiation on cellular target molecules may lead to various functional consequences. These functional effects can be broadly categorized into cell death, repair, cell cycle effects, altered gene expression, modification of signal transduction, mutagenesis, and genomic instability (Fig. 3.2). These categories are not exclusive.

Cell Death

Among the functional consequences of ionizing radiation, cell death is the most important one for radiation oncology. Cells can die in several ways⁹⁰—by apoptosis, mitotic catastrophe, senescence, necrosis, and autophagy (Table 3.2). Most important for the effect of radiotherapy of solid tumors is mitotic catastrophe, which is caused by lethal chromosome damage.⁹¹ After irradiation, cells can pass through one or few mitotic cycles before missegregation of chromosomes or cell fusion leads to the loss of their replicative potential (or clonogenicity). Often micronuclei, containing nonrepaired chromosome fragments, can be detected. Those micronuclei, and hence essential genetic information, will be lost during the following cell cycle, which results in cell death. Frequent multinucleate giant cells reflect a radiation-induced failure of cytoplasmatic separation on cell division.

Neoplastic hematopoietic or lymphatic cells often die from radiation-induced apoptosis via the intrinsic, caspase 9–dependent pathway. Two different forms of radiation-induced apoptosis can be distinguished—early or premitotic versus late or postmitotic.⁹² Early apoptosis is p53 dependent, occurs within few hours after irradiation before the cells enter mitosis, and is primarily a consequence of DNA damage. Early apoptosis represents a distinct mode of radiation-induced cell death. In contrast, secondary apoptosis occurs after mitosis and is one of several possible manifestations of radiation-induced lethal chromosome aberrations. In most cancers, particularly in solid tumors, apoptosis appears not to be the main mechanism of radiation-induced cell death.⁵³ No clear evidence exists that either apoptotic index or levels of p53, Bcl-2, or other Bcl-2 family members are predictive of the response of solid tumors to radiotherapy. For example, overexpression of Bcl-2 was shown to significantly decrease the apoptotic fraction in response to irradiation. However, this did not translate into a change of clonogenic cell survival after irradiation.⁹³

Radiation-induced senescence plays an important role for development of normal tissue damage—for example, fibrosis⁹⁴—but occurs also in response to nonlethal stress as in tumor cells.⁹⁵ Cells survive and are metabolically active but lose their replicative potential. Necrosis is an unregulated process of cell destruction by the release of intracellular components. This is usually the consequence of pathophysiologic conditions such as ischemia and inflammation.⁹⁰ So far no distinct pathway has been described that directly leads to cellular necrosis after clinically relevant doses of irradiation. However, it is well known that tumors often show massive necrosis after neoadjuvant radiotherapy or radiochemotherapy, which in some tumors correlates with improved prognosis.⁹⁶^,⁹⁷ It is likely that this radiation-induced induction of necrosis is explained by several factors, including mitotic catastrophe of tumor cells and the effects of irradiation on tumor vessels leading to changes in the microenvironment, which consequently cause cell death.

Autophagy is a form of nonapoptotic and nonnecrotic cell death that is related to lysosomal degradation of proteins and cell organelles that are then utilized for the production of new cells.⁹⁰ This mode of programmed cell death is triggered by growth factor withdrawal, differentiation, and developmental stimuli. Although the molecular regulation is not completely understood, recent data suggest that a high rate of autophagy contributes to radiation resistance⁹⁸and that autophagy is regulated through different hypoxia-dependent pathways, thereby facilitating survival during metabolic stress.⁹⁹

Recognition of Radiation-Induced DNA Damage

DNA damage, in particular DSBs, is sensed by different proteins that trigger an ataxia telangiectasia mutated (ATM)–dependent or, in some cases, ataxia telangiectasia and Rad3-related (ATR)–dependent signaling cascade¹⁰⁰ (Fig. 3.3). ATM activation requires the telomeric protein TRF2 and the MRN complex consisting of Rad50, meiotic recombination protein 11 (Mre11), Nijmegen breakage syndrome protein 1 (NBS1), mediator of DNA damage checkpoint protein-1 (MDC1), and 53BP1.¹⁰¹ In addition to proper activation of downstream DNA-repair proteins, histone H2AX molecules need to be phosphorylated in the vicinity of the DSB. All of these proteins assemble at the site of the breaks and presumably control the choice of one of several repair pathways. In addition to its role in repair, ATM is involved in the regulation of multiple cell cycle checkpoints (G1/S, S, G2/M) after DNA damage.¹⁰²ATM, ATR, and DNA-dependent kinase-catalytic subunit (DNA-PK_CS) phosphorylate p53 and a number of proteins involved in cell cycle delay, apoptosis, and induction of DNA repair. ATR appears to be important for sensing ultraviolet-related and other types of bulky lesions, as well as damage that induces a replication block, while ATM seems to be the sensor for DSB induced by ionizing radiation.¹⁰⁰ ATR may serve in some cases as a backup for ATM.

FIGURE 3.3. Simplified pathways of radiation-induced DNA damage recognition. ATM, ataxia telangiectasia–mutated; ATR, ataxia telangiectasia and Rad3–related; MRN, Mrc 11, Rad50, NB51 complex; PK, protein kinase.

DNA Repair

Genome integrity is essential to the survival of cells and organisms. It is estimated that about 10⁴ DNA lesions occur in a single human cell every day.¹⁰³ Most of the damage is caused by endogenous sources such as oxygen free radicals, replicative errors, and spontaneous desaminations. To cope with the plethora of permanent damage initially, a repair system was developed during evolution that also acts on external challenges such as radiation or chemical DNA damage. To enable repair of massive DNA damage, the cells stop proliferation. This may prevent replication or segregation of damaged genetic material. If repair is not possible, the cells either die or, in case of survival, may propagate mutated DNA (Fig. 3.2). When the DNA replication machinery meets damaged DNA, replication may continue despite the damage by translesion synthesis, which requires DNA polymerase with low fidelity. Different mechanisms are involved in DNA repair.¹⁰⁰^,¹⁰⁴

Base Excision Repair

The mechanism of base excision repair (BER) is responsible for the repair of various kinds of base damage (abasic sites, oxidized bases, deaminated bases, and alkylated bases) and SSB. Base damages and SSB are the most frequent types of DNA damage after irradiation.¹⁰⁵^,¹⁰⁶ The major BER pathway is the short-patch pathway, which involves excision of only one base. The minor pathway—the long-patch repair—excises 2 to 10 nucleotides. In the short-patch pathway, DNA glycosylases recognize damaged bases and excise them from DNA. Apurinic/apyrimidinic endonuclease 1 (APE1 synonyms: HAP-1, redox effector factor 1) hydrolyzes the phosphodiester bond 5′ to the abasic site. Alternatively, an AP-lyase cleaves the 3′ sugar-phosphate bond. In both cases, the sugar residue is removed by either a 5′- or a 3′-phosphodiesterase. Polymerase β (Pol-β) incorporates a single nucleotide into the gap, and, finally, the nick in the DNA is sealed by DNA ligase III, which interacts with Pol-β. In this process, x-ray repair cross-complementing protein (XRCC1) interacts with several enzymes in this pathway and regulates their activity.¹⁰⁷ For the long-patch repair, the nick produced by AP-endonuclease or AP-lyase is extended to a gap 2 to 10 nucleotides by either a 5′- or a 3′-exonuclease, that is, exonuclease function of polymerase δ or (Pol-δ or ), or by removal of a flap end by flap-end endonuclease-1 (Fen-1). Pol δ or and associated replication factors (proliferating cell nuclear antigen and replication factor C) then fill the gap. After the synthesis, DNA ligase I or, less frequently, ligase III, seals the nick.¹⁰⁸

FIGURE 3.4. Mechanisms of DNA double-strand break repair in mammalian cells.

DSB Repair

Spontaneous DSBs occur either on topoisomerase failure during recombination and are forced by replication errors or are due to thermodynamic fluctuation. In germ cells, DSBs are produced during meiotic crossover in T and B lymphocytes during a sik-specific DNA recombination of variable diversity and joining genes (VDJ) on maturation of T cell receptors and antibodies. Radiation-induced DSBs are, despite their relatively low induction frequency (40 per Gy per cell), biologically much more important than base damage and SSB. Two major pathways—homologous recombination (HR) and nonhomologous end joining (NHEJ)—have evolved to repair DSB¹⁰⁹^,¹¹⁰ (Fig. 3.4). Whereas NHEJ occurs in all phases of the cell cycle, HR is particularly important in the S/G2 transition of the cell cycle.

HR is a slow, high-fidelity repair pathway. Regions of DNA homology—usually the sister chromatid—are used as the template. During HR, activated ATM recruits endonucleases that process broken ends, which ultimately creates single-stranded 3′ ends. For the processing, the MRN complex is required. In concert with BRCA1, BRCA2, and Rad51 paralogues (XRCC2, XRCC3, Rad51B, Rad51C, Rad51D, Rad52, Rad54), the Rad51 protein binds to single-stranded DNA with the aid of replication protein A and searches for a homologous sequence on the sister chromatid. On strand invasion, Rad51 enables the formation of a temporary triple helix. Once the complementary strands have paired, the 3′ end of the damaged DNA will be elongated by polymerases (not yet identified) beyond the position of the former DSB. When the 3′ single strand of the second end of the DSB also invades the structure, a quadruple-helix is formed called a Holliday junction, which can be extended in both directions. After the gap has been safely bridged (usually about 50 base pairs will be copied), the Holliday junction is resolved, and the remaining nicks are sealed by a DNA ligase (not yet identified).

NHEJ is a fast but error-prone, and thus potentially mutagenic, repair pathway that rejoins DNA ends, usually after removal of a limited number of base pairs. NHEJ is initiated by the Ku70/Ku80 heterodimer, which binds to DNA ends and recruits the DNA-PK_CS. DNA-PK_CS can phosphorylate a variety of repair proteins such as Ku, x-ray cross-complementation protein 4 (XRCC-4), Artemis, p53, or replication protein A. However, only the autophosphorylation has been identified as being essential for repair. Artemis, in concert with DNA-PK_CS, trims the DSB ends for subsequent processing. After release of DNA-PK_CS from the DNA, the end will be bridged by the complex of ligase IV/XRCC-4/XRCC-4-like factor (XLF), which also performs the final ligation step. It has been observed that NHEJ can, to some extent, occur in the absence of several core proteins. This gave raise to the idea of a backup pathway that only operates when the DNA-PK–dependent NHEJ fails.¹¹¹ Recent data suggest that, besides the genuine repair proteins, the tumor suppressor p53 is involved in controlling the repair. P53 appears to suppress both HR and NHEJ in case error-free repair is not possible and thereby reduces the mutagenic risk of error-prone repair.¹¹²

Despite our molecular knowledge about DSB repair, the impact of the chromatin organization on DSB induction and repair has only recently been recognized.¹¹³^,¹¹⁴^,¹¹⁵^,¹¹⁶ Chromatin is organized as euchromatin and heterochromatin, representing loose and condensed DNA areas, respectively. While DSBs in euchromatin are easily accessible for repair proteins, DSBs in heterochromatic DNA regions need to be moved to less condensed areas for efficient repair.¹¹⁴^,¹¹⁶ Key molecules involved in this differential chromatin-dependent processes are ATM in association with heterochromatic marker proteins like KAP-1 and 53BP-1 and certain types of histones.¹¹³^,¹¹⁵

Radiation-Induced Cell Cycle Delay

It has long been recognized that radiation-induced DNA damage is associated with delay in the cell cycle, which has been interpreted as allowing additional time for the cells to repair. Most recent findings show that DSBs can be found in all cell cycle phases, suggesting incomplete repair before entering the next phase. For example, tracking of radiation-induced γ H2AX foci, representing DSBs, revealed transfer of DSBs induced in the G1 cell cycle phase to daughter cells.¹¹⁷

G1 Phase

The G1 cell cycle checkpoint prevents damaged DNA from being replicated and is the best understood checkpoint in mammalian cells.¹¹⁸ Radiation-induced G1 phase cell cycle arrest is regulated by p53 (Fig. 3.5). Loss of p53 function, which is found in the majority of tumors, leads to a lack of G1-phase arrest. Instead, these cells exert a dose-dependent blockage in the G2 and M phases of the cell cycle. Central to the G1 checkpoint is the accumulation and activation of the p53 protein, which is controlled by the ATM and ATR kinases. These kinases, together with DNA-PK, are activated by and recruited to radiation-induced DNA lesions. Physiologically, p53 expression levels are low due to interaction with its negative regulator MDM2, which targets p53 for nuclear export and proteasome-mediated degradation in the cytoplasm.¹¹⁹ Following radiation-induced DNA damage, ATM activates the downstream cell cycle checkpoint kinase Chk2 by phosphorylation at position Thr68,¹²⁰ which in turn phosphorylates amino acid residue Ser20 of p53. This results in a pronounced tetramerization, activity, and stability. The p53-Ser20 phosphorylation inhibits p53/MDM2 interaction, resulting in p53 accumulation. Moreover, ATM exerts p53 stability by directly phosphorylating MDM2 on Ser395.¹²¹ Although it allows maintenance of MDM2/p53 interaction, this event prevents p53 nuclear export to the cytoplasm for degradation. In vitrostudies showed Ser20 phosphorylation of p53 by the ATR-related cell cycle checkpoint–dependent kinase Chk1.¹²² Transcriptional transactivation activity is mediated via Ser15 phosphorylation of p53.¹²³ Both ATM and ATR are able to directly phosphorylate this residue in response to irradiation. P53 target genes include several genes that are involved in the DNA damage response (e.g., MDM2, GADD45a, p21^Cip1). Accumulation of the cyclin-dependent kinase inhibitor p21^Cip1 blocks G1/S-phase progression by binding to the cyclinE/cdk2 complex, reducing cdk2 activity, which has an important function in phosphorylation of pRB.

S Phase

After irradiation, the rate of DNA synthesis is decreased via ATM- and NBS1-dependent pathways.¹²⁴ Radiation-induced DNA damage activates ATM for Thr68 phosphorylation of Chk2.¹²⁵ Activated Chk2 targets CDC25A phosphatase for ubiquitination. As a result of CDC25A degradation, inhibitory phosphorylations of Cdk2 reside at Thr14 and Tyr15. The Cdk2/cyclin E and Cdk2/cyclin A complexes remain inactive, which prevents completion of DNA synthesis and G2 entry. Alternatively, radiation-activated ATM phosphorylates several downstream substrates, including BRCA1 at Ser1387, NBS1 at Ser343, and SMC1 at Ser957 and Ser966.¹²⁵^,¹²⁶ ATR phosphorylates the cell cycle checkpoint kinase Chk1 at Ser317 and Ser345. Subsequently, Chk1 phosphorylates CDC25A, which results in cytoplasmic sequestration and thereby in inhibition of S–G2 transition.¹²⁷

FIGURE 3.5. Pathways involved in radiation-induced G1/S and G2 cell cycle blocks. ATR, ataxia telangiectasia and Rad3–related; ATM, ataxia telangiectasia–mutated; IR, ionizing radiation.

G2 Phase

In contrast to the G1 checkpoint, all mammalian cells, normal or transformed, undergo cell cycle arrest in G2 after radiation-induced damage.¹²⁸ The G2 cell cycle checkpoint is the essential final determinant allowing cells to divide. The entry into mitosis is regulated by the activity of the cyclin-dependent kinase Cdk1¹²⁹ (Fig. 3.5). Phosphorylation of Thr14 and Tyr15 of Cdk1 blocks cell cycling into G2. These phosphorylations are removed by the phosphatase CDC25C. After irradiation, ATR and ATM activate the downstream checkpoint kinases Chk1 and Chk2, which phosphorylate CDC25C at Ser216.¹³⁰ This results in binding of 14-3-3 proteins. The CDC25C/14-3-3 protein complex is translocated into the cytoplasm for sequestration. As a consequence of this CDC25C degradation, Cdk1 remains phosphorylated, thereby preventing entry into mitosis. ATM appears to be more dominant at the early stage of G2/M checkpoint activation, whereas ATR seems to contribute mainly to sustained checkpoint events.¹³¹

Radiation-Induced Gene Expression

Radiation may modify gene expression.¹³² Many of the transcriptionally activated proteins have been shown to be centrally involved in the pathogenesis of radiation damage or to modulate the effect of radiation on tumor cells. Early responses may occur within hours after irradiation and involve mostly transcription factors such as the proto-oncogenes jun, fos, junB, and early growth response gene 1 (Egr1).¹³³ Sustained activation of early radiation response genes such as NFκB may contribute to late radiation damage.¹³⁴ Other examples of radiation-induced genes that may occur early and/or late after irradiation include TGF-β, TNF-α, bFGF, PDGF, and interleukin 1 (IL-1).

Induction of jun is partly mediated by protein kinase C¹³³ and by reactive oxygen intermediates.¹³⁵ In addition to transcriptional activation, prolongation of the half-life of jun has also been observed.¹³⁶ Jun and fos form a heterodimer that represents the transcription factor AP-1. This transcription factor is a central regulator of cell proliferation, differentiation, and death.¹³⁷ For example, activation of AP-1 results in transcriptional suppression of MAPK phosphatases.¹³⁸ Furthermore, AP-1 appears to be an important regulator of the transcription of other radiation-induced genes, such as in the early transcription of TGF-β.¹³⁹ Redox-dependent DNA-binding activity of AP-1 is regulated by the DNA repair protein Ref-1.¹⁴⁰

Gene expression and DNA-binding activity of NFκB are induced soon after ionizing radiation.¹⁴¹ NFκB is a sequence-specific DNA-binding protein complex that binds to DNA as a dimer. Five mammalian proteins have been identified that belong to this family: NFκB1 (p50 and its precursor p105), NFκB2 (p52 and its precursor p100), c-Rel, RelA (p65), and RelB.¹⁴² The most frequently occurring dimer is p50/p65. In the nonactivated state, this dimer is retained in the cytoplasm by binding to its inhibitor IκB (inhibitor of NFκB). On activation of the NFκB pathway, IκB is phosphorylated by a kinase complex named IκB kinase (IKK), consisting of the three subunits—α, β, and γ. According to its multiple functions, NFκB can also be activated by a variety of agents, like membrane receptors such as TNFR or Toll-like receptors and oxidative stress. Radiation-induced activation of NFκB is mediated by IKK.¹⁴³This response does not depend on a nuclear signal, as it also occurs in enucleated cells.¹⁴⁴ However, it has been shown that ATM is required for chronic activation of the transcription factor NFκB.¹⁴⁵ NFκB acts in an antiapoptotic way and mediates radioresistance.¹⁴⁶

Radiation exposure induces immediate and sustained TGFβ1 gene expression, as well as activation of this cytokine.¹⁴⁷ On transcriptional activation TGF-β is produced as an inactive latent form and secreted. Latent TGF-β is stored in the extracellular matrix and proteolytically activated in irradiated tissues.¹⁴⁸ Active TGF-β1 binds and activates the TGF-β1 type I receptor, resulting in TGF-β1–dependent gene expression, inducing, for example, p21, p27, collagen, and TIMP.¹¹^,¹³⁹ This is most likely responsible for the cellular effects of TGF-β, such as modulation of proliferation, differentiation, and radiation sensitivity of fibroblasts, and for the biochemical events (e.g., collagen deposition, characteristic of radiation-induced fibrosis). In addition, TGF-β secreted by tumor cells on irradiation might contribute to the development of radiation-induced fibrosis.¹⁴⁹ Intervention of TGF-β–mediated effects (e.g., by TGF-β1–neutralizing antibodies and superoxide dismutase) offers a promising strategy to prevent radiation-induced fibrosis. Aside from fibrosis, TGF-β has been demonstrated to reduce latency, promote aggressive tumor growth, and increase estrogen receptor–negative breast cancers.¹⁵⁰ Moreover, the radiosensitivity of cancer cells can be enhanced by TGF-β–directed treatments.¹⁵¹^,¹⁵²

TNF-α belongs to the group of proinflammatory cytokines involved in radiation-induced normal tissue damage, such as pneumonitis and lung fibrosis. Immediately after irradiation, TNF-α is transcriptionally upregulated and released by the bronchiolar epithelium. TNF-α enhances phagocytosis and cytotoxicity by neutrophilic granulocytes and modulates the expression of other cytokines such as IL-1 and IL-6.¹⁵³ Experimental data show that also tumor lines, particularly pediatric sarcomas, may produce large quantities of bioactive TNF-α after irradiation. This may be of potential importance for tumor response and for normal tissue reactions after radiotherapy.¹⁵⁴

Induction of genes by ionizing radiation has been experimentally exploited for therapy. For example, the early-response gene Egr1 was inserted upstream to TNF-α, which may act as a radiosensitizer. This provides a strategy for spatial and temporal control of the biologic effect by radiotherapy.¹³²^,¹⁵⁵

Radiation Effects on Signal Transduction

Ionizing radiation may activate intracellular signaling.¹⁵⁶ The general mechanisms of activation involve the dose-dependent production of ROS and reactive nitrogen species (RNS), which stimulate, for example, cytoplasmic protein kinases, phosphatases, and cell membrane receptors or disturb, for example, lipid and protein metabolism.¹⁵⁷ Many signaling pathways are simultaneously activated in a dose- and cell type–dependent manner, which may contribute to different radiation responses in different cell types. Cooperative and mutual cross-talk occurs between parallel and upstream and downstream signaling routes.¹⁵⁸ Examples of important intracellular signaling pathways and their modulation by ionizing radiation are discussed in the following paragraphs.

EGFR-Mediated Signaling

The EGFR family belongs to the group of RTKs and consists of four different single receptors (EGFR/ErbB-1, HER2/ErbB-2, HER3/ErbB-3, and HER4/ErbB-4) that are dimerized after ligand binding to the extracellular domain.¹⁵⁹In addition to activation through its natural ligands such as EGF, TGF-α, or amphiregulin and transactivation by cell adhesion molecules like integrins or immunoglobulin-like receptors,¹⁶⁰ ionizing radiation is able to stimulate EGFR.¹⁶¹ Radiation doses of 1 to 2 Gy activate the EGFR and its downstream signaling cascades with similar efficiency as physiologic EGF concentrations of 0.1 to 1 nM. Radiation-dependent production of ROS/RNS seems to play a critical role in EGFR activation and downstream signaling via MAPK.¹⁵⁷^,¹⁶² It was shown that protein tyrosine phosphatases contain ROS/RNS–sensitive cysteine residues at a site essential for phosphatase activity. Thus, the radiation-dependent activation of EGFR is likely to be controlled by ROS/RNS–regulated protein phosphatases. In addition, paracrine and autocrine activation of the EGFR can also result from radiation-induced release of TGF-αfrom irradiated cells or by release of growth factors stored in the extracellular matrix as result of activation of matrix-degrading proteases.¹⁶³^–¹⁶⁵

EGFR signaling via Ras-Raf-MAPK after irradiation has been implicated in increased proliferation,¹⁶⁶ which corresponds to observations that EGFR overexpression is associated with repopulation during fractionated irradiation.^167,¹⁶⁸^–¹⁶⁹ In addition, EGFR-dependent signaling via PI3K/Akt has been associated with increased cellular survival and cell cycle progression.¹⁷⁰^,¹⁷¹ Another EGFR downstream pathway represents the c-Src–mediated activation of STAT3. STAT3 is involved in the TGF-α–mediated autocrine growth¹⁷² and contributes to the regulation of angiogenesis by production of VEGF.¹⁷³ After radiation-induced activation, the EGFR is internalized and may function as a nuclear transcription factor.¹⁷⁴ In addition, internalized EGFR can activate DNA-PK. Pharmacologic inhibitors can block EGFR signaling at different levels and thereby influence several mechanisms of radioresistance, such as DNA repair, repopulation, antiapoptotic signaling, and tumor hypoxia.¹⁸ The specific mutational status of the cells—for example, Ras mutations¹⁶⁴^,¹⁷⁵—and the class of drugs¹⁷⁶^,¹⁷⁷ may critically modify effects of EGFR inhibition on radiation response. The concept of EGFR inhibition to improve outcome of radiotherapy has been proven recently in a phase III clinical trial.¹⁷ However, considerable intertumoral heterogeneity has been observed, which is mechanistically only partly understood.¹⁹ An important variant of the EGFR for radiation oncology is the constitutively active, truncated EGFRvIII, which is coexpressed with EGFR in a significant proportion of solid tumors.¹⁷⁸ EGFRvIII lacks the ability of EGF binding due to a deletion of the NH₂-terminal domain but, like wild-type EGFR, can be activated by irradiation.¹⁷⁹ Evidence suggests that EGFRvIII has altered signaling properties compared to normal EGF receptor and represents a cancer cell–specific target.¹⁷⁸ Experimental data indicate that activation of EGFRvIII leads to more-pronounced cytoprotective responses to radiation than wild-type EGFR.¹⁷⁹

Ras Signaling

Mutant, constitutively active Ras, an important component of the MAPK pathway, has been found in many types of human cancers. Farnesyltransferase inhibitors (FTIs) can effectively block Ras-mediated signal transduction.¹⁸⁰Application of FTI renders cells more sensitive to ionizing radiation. Combination of FTI with radiotherapy is under preclinical and clinical investigation.³⁸^,¹⁸¹

PDGF-Mediated Signaling

Radiation-induced autocrine and paracrine PDGF signaling plays an important role in fibroblast and endothelial cell activation and proliferation in vitro.¹⁸² Combination of irradiation with PGDFR tyrosine kinase inhibitors resulted in decreased clonogenic survival of endothelial cells and fibroblasts in vitro.¹⁸² Recent data suggest that inhibition of PDGFR signaling may attenuate development of radiation-induced pulmonary fibrosis.¹⁸³

VEGFR-Mediated Signaling

Transmembrane receptors for VEGF and related ligands include VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), VEGFR-3 (Flt-4), neuropilin-1, and neuropilin-2.¹⁸⁴ On irradiation, VEGF is upregulated via the EGFR/STAT signaling and released by tumor cells and exerts, via VEGFR/PI3K/Akt signaling, prosurvival stimuli on tumor and endothelial cells.¹⁸⁵ Upregulated VEGFR expression has been found after ionizing irradiation.¹⁸⁶^,¹⁸⁷ Besides normalizing the tumor micromilieu,¹⁸⁸ inhibition of radiation-induced VEGF/VEGFR signaling represents the rationale for combining anti-VEGF strategies with radiotherapy, which is under preclinical and clinical investigation.¹⁸⁹

Cyclooxygenase–Mediated Signaling

The rate-limiting enzyme in the synthesis of prostaglandins is cyclooxygenase (COX). Two isoforms exist, COX-1 and COX-2. COX-2 is typically not expressed or is expressed at relatively low levels in normal tissues but overexpressed in 40% to 80% of cancers of the lung, colon, head and neck, breast, prostate, brain, and pancreas.¹⁹⁰ Among other stimuli, irradiation has been shown to upregulate expression of COX-2 in tumors and normal tissues.¹⁹¹^,¹⁹² COX-2 expression correlates with worse outcome after radiotherapy in patients.¹⁹³^,¹⁹⁴ Specific inhibition of COX-2 has been demonstrated to enhance radiation responses of tumor cells in vivo and in vitro.¹⁹⁵ Several mechanisms, including reduced angiogenesis, increased apoptosis, and immune responses, have been implicated in the radiosensitizing effects of COX-2 inhibitors. Recent experimental data suggest that inhibition of DNA repair by COX-2 inhibitors via downregulation of Ku70, and thereby inhibition of DNA-PK_CS, contributes significantly to the radiosensitizing effects.¹⁹⁶ Furthermore, COX-2 inhibitors appear to attenuate radiation gene expression via inhibition of NFκB.¹⁹⁶ Ongoing early clinical trials in combination with radiotherapy indicate feasibility of this approach,¹⁹⁷^,¹⁹⁸ although conflicting data exist¹⁹⁹ and efficacy at least for combination with radiochemotherapy in unselected non–small cell lung cancer patients may be questionable.²⁰⁰

Integrin-Mediated Signaling

Interactions between cells and ECM proteins are facilitated mainly by the integrin family of cell adhesion molecules.²⁰¹ Besides the influence of growth factors and cytokines, these interactions add a further facet to the network of microenvironmental factors that modulate the cellular behavior on exposure to ionizing radiation (Fig. 3.6). Chemotherapeutic compounds, as well as ionizing radiation, showed less cytotoxic efficacy in cells adherent to ECM compared to cells growing in suspension or on plastic surfaces.²⁰² Resistance-promoting effects by integrin-mediated adhesion to ECM were found in cell lines from solid tumors of the lung,²⁰³^,²⁰⁴ breast,²⁰⁵ liver,²⁰⁶ colon, pancreas,²⁰²^,²⁰⁷^,²⁰⁸ ovary,²⁰⁹ prostate,²¹⁰ brain,²¹¹ and leukemia cells.²¹² Certain integrins seem to communicate increased radiation and drug resistance, which is cooperatively influenced by RTKs like EGFR.²¹³ For example, α5β1 integrin, the “classic” fibronectin receptor, acts as a major antagonist of cell death induced by doxorubicin or melphalan in multiple myeloma,²¹⁴ by paclitaxel in breast cancer or small cell and non–small cell lung cancer,²¹⁵^,²¹⁶ or by cisplatin or mitomycin C in non–small cell lung cancer.²¹⁵ Additional studies underscored the important role of β1 integrins in promoting resistance against ionizing radiation on the basis of signaling events via the adaptor proteins paxillin and p130Cas to the survival-regulating protein kinase JNK.²¹⁷^,²¹⁸ Further regulatory cytoplasmic cascades downstream of β1 integrins that exert survival advantage on treatment with genotoxic agents included regulation of the Akt1/FoxO cascade by the adaptor protein PINCH1,²¹⁹ the proapoptotic proteins Bim and Bax,²²⁰^,²²¹ and the antiapoptotic Bcl-2–like proteins or Bcl-2/Bax.²¹² In addition to regulating cell survival, cell–matrix interactions affect radiation-induced cell cycle arrest, that is, adhesion of cells to matrix proteins prolongs the G1 and G2 cell cycle blockage in parallel with enhanced activation of DNA repair pathways involving the checkpoint kinases Chk1, Chk2, and Cdk1, p53, and diverse cyclins.²¹⁵^,²²²^–²²⁴

Following radiation exposure, the expression of several integrin subunits, including β1, β3, α5, and αv, is upregulated in human skin and lung fibroblasts,²⁰² endothelial cells, and keratinocytes,²²⁵ as well as in tumor cells of the colon, hematopoietic cells,²²⁶ prostate,²²⁷ lung,²²⁸ brain,²²⁹ and melanomas.²⁰² Generally, integrin-mediated adhesion to matrix proteins confers chemoresistance and radioresistance.²¹²^,²¹³^,²³⁰

FIGURE 3.6. Cross-talk between receptor tyrosine kinase (RTK) and integrin-mediated signaling.

Mutagenesis and Genomic Instability

Cells may survive radiation despite unrepaired or misrepaired DNA damage, that is, with mutations or chromosomal aberrations. These may remain silent or result in radiation-related secondary primary cancers or, as germline mutations, cause hereditary disease.²³¹^–²³³ In addition to directly induced mutations, radiation may induce a heritable, genome-wide process of instability (i.e., genomic instability) that leads to an enhanced frequency of genetic changes occurring among the progeny of the original irradiated cell, which is transmissible over many generations of cell replication.²³⁴ Whereas most mutations induced directly by radiation involve loss of large parts of the tested gene, leading to a loss of heterozygosity, most mutations resulting from radiation-induced genomic instability involve point mutations and small deletions.²³⁴^,²³⁵ While some studies, particularly on thyroid cancer in children after the Chernobyl fallout, suggest a nonrandom pattern of chromosomal damage, generally no fingerprint alterations have been identified that would unequivocally indicate radiation-induced cancer.²³³

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