- Email: [email protected]
Biology of cancer
CANCER BIOLOGY
What’s new ?
Biology of cancer
• Gene therapy approaches to the treatment of cancer have been developed, based on the expression of wild-type versions of mutated tumour-suppressor genes
Ian R Hart
• Several specific tyrosine kinase inhibitors have been introduced into clinical practice • The first successful trial of anti-angiogenic treatment has been reported, in metastatic colorectal cancer Cancer is a spectrum of genetic diseases resulting from various mutations in specific genes. These mutations, which may either abrogate gene function or increase gene function (by causing constitutive activation), drive cells toward the unregulated and irreversible cellular proliferation that is the hallmark of cancer. Over the last 10–15 years, considerable progress has been made in defining and understanding these changes at the molecular level. Development of novel therapeutic approaches derived from this increased understanding has been slower, but it is hoped that elucidation of the biochemical pathways involved in tumour development, progression and metastasis will identify novel targets against which agents can be developed to regulate these processes. Some are already entering clinical trials. This contribution focuses on the areas of cancer biology that appear to offer the greatest opportunity for short-term development of novel therapeutic agents, and shows how increased understanding of the pathogenesis of malignancy can provide a means of developing such treatments.
lular DNA of animals, where they did not operate as cancer genes. It appears that the viral (v-onc) genes were acquired by viruses from normal cells during viral evolution and were integrated into the virus. Subsequently, these genes caused tumorigenicity when expressed in virus-infected cells. Tumorigenicity could result from
Experiment showing that DNA contains genes capable of conferring a tumorigenic phenotype on putatively normal cells a High molecular weight tumour DNA precipitated with calcium phosphate
b Normal recipient cells (murine 3T3 fibroblasts)
Development of cancer Dominant-acting oncogenes Epidemiological studies have shown that about 80% of cancers result from environmental factors. These factors lead to gradual acquisition of a series of mutations in target cells, resulting in the progression of a normal phenotype to a neoplastic one. These mutations often confer a survival benefit on the cells, leading to preferential growth of a new focus of mutant cells that can become a target for further mutations. In the early 1980s, a series of elegant experiments involving transfer of only tumour-derived DNA to recipient cells demonstrated that the transformed phenotype could be acquired in a dominant manner (Figure 1). It became apparent that many of the DNA sequences causing these changes in recipient cell transformation status were already known – they had been identified as oncogenes (onc), which were additional sequences within the viral genomes of RNA tumour viruses (retroviruses). These onc genes were shown to be almost identical to sequences in the normal cel-
c Focus formation
d Foci grow as tumours in mice
High molecular weight DNA isolated from tumour is precipitated with calcium phosphate and transfected into normal, non-tumorigenic recipient cells of the murine 3T3 fibroblast line. In the presence of transforming DNA, the 3T3 cells pile up and form evident foci in the Petri dish. Inoculation of such cells into recipient mice leads to the formation of rapidly growing fibrosarcomas. Alternatively, when these transformed cells are plated in soft agar they grow in an anchorage-independent manner.
Ian R Hart is Professor of Tumour Biology at Barts and The London, Queen Mary’s School of Medicine and Dentistry, London, UK. He qualified as a veterinary surgeon from the University of Bristol, and undertook research at the University of Bristol and in the USA. His research interests include the role of cell adhesion in tumour spread.
MEDICINE
e Anchorage independent in soft agar
1
1
© 2004 The Medicine Publishing Company Ltd
CANCER BIOLOGY
over-expression of the encoded product, or from differences in sequence between the viral and cellular homologues – the transforming DNA identified by transfection techniques (using DNA isolated from cancers) contained comparable alterations. These oncogenes appear to actively cause increased cell proliferation. They are thought to lie within the biochemical pathways driving cellular proliferation, and could: • be members of the tyrosine kinase family (responsible for phosphorylating downstream proteins) • have GTPase activity (i.e. bind guanosine triphosphate and convert it to the diphosphate and monophosphate forms), which is involved in signal transduction cascades (transmission of signals from cell surface to nucleus) • be related to growth factors or growth factor receptors • regulate gene expression by binding to DNA, as a result of their nuclear localization.
coli gene, p53, and the breast cancer 1 (BRCA1) and 2 (BRCA2) genes. In normal organisms, cell proliferation is balanced by regulated cell loss, often through apoptosis (programmed cell death). Many tumour-suppressor genes are involved in cell proliferation, in genetic stability and in cell death, and they are often mutated in cancer; such mutations in these genes generally ablate the ability to cause apoptosis and result in increased cell numbers. Implications for therapy Understanding of the biochemical pathways that drive cellular proliferation may lead to development of specific inhibitors; for example, tyrosine kinase inhibitors may block phosphorylation of proteins involved in the signalling that induces cell proliferation, whereas inhibitors of GTPase could prevent activity of oncogenes through this pathway. Alternatively, if mutated tumour-suppressor genes can be replaced, cellular proliferation can be suppressed and apoptosis in the target cell population may increase. This concept underlies gene therapy protocols aimed at re-expressing tumoursuppressor genes (e.g. p53) specifically in tumour cells. Recent reports of an international phase II/III trial of p53 gene delivery as a first-line treatment in ovarian cancer were disappointing, but in such gene-therapy protocols the major limiting factor is almost certainly the efficiency of gene delivery. The best example of a novel approach to cancer therapy based on the role of oncogenes in tumour biology is the use of a monoclonal antibody that recognizes the HER2-neu (c-erbB2) oncogene. HER2-neu encodes a transmembrane tyrosine kinase receptor with extensive homology to the epidermal growth factor
Tumour-suppressor genes Tumour-suppressor genes were first suggested in studies of rare inherited cancer syndromes. In familial retinoblastoma, for example, a genetic change was inherited through the germ line, but mutation or ablation of the other allele on the homologous chromosome was required for tumour development (Figure 2). In sporadic cases, independent mutational events in both homologous genes are required; as a result, sporadic retinoblastoma is less common than the familial form. Many tumour-suppressor genes have now been identified, including the retinoblastoma gene, the adenomatous polyposis
Inherited
The role of herceptin in preventing HER2/neu dimerization and subsequent generation of a signal transduction event
Sporadic
HER2/neu receptor Rwt
Rdel
Rwt
Ligand binding
Blockade
Rwt
Step 1
Herceptin Cell membrane
m
R
R
del
wt
R
R
m
P
P
Step 2 P P
Rm
Rm
P P
Inactive receptor monomer is activated by ligand binding, which brings together the monomers to form active dimers. Dimerization results in transient activation and phosphorylation of specific tyrosine residues within the intracellular domain. These phosphorylation events initiate signals that result in changes in gene expression and are associated with growth of the tumour. The herceptin antibody blocks binding of the ligand to the receptor.
In sporadic cancers such as retinoblastoma, two mutations are required to eliminate the retinoblastoma gene. In inherited cases, a mutated or deleted copy is inherited in the germ line and thus only one event is needed in any cell to eliminate both copies of the gene. 2
MEDICINE
P P
3
2
© 2004 The Medicine Publishing Company Ltd
CANCER BIOLOGY
receptor (Figure 3). Ligands that bind to the extracellular domain of this receptor induce dimerization and internalization, and activate intracellular tyrosine kinase activity. Gene amplification of HER2neu has been documented in up to one-third of breast cancers, and a similar proportion have increased protein levels in the absence of gene amplification. Herceptin is an anti-HER2 antibody that has been tested in a phase III clinical trial. Alternatively, rather than attempting to block the extracellular receptor, it might be possible to develop small molecules that inhibit the downstream sequelae of binding of the appropriate ligand to the receptor. This approach has been used with several tyrosine kinase inhibitors including ZD1839, which is proposed to inhibit signalling from epidermal growth factor receptors. The most successful agent of this type is perhaps ST1-571 (imatinib), which efficiently inhibits the Bcr-Abl oncoprotein. This tumour-specific tyrosine kinase is produced through fusion of the ABL and BCR genes, which occurs as a result of chromosomal translocations (the Philadelphia chromosome) in chronic myeloid leukaemia.
veloping tumour mass. New vessels are induced to form as a result of the release of a variety of angiogenic peptides, which may be produced by the neoplastic cells or by host inflammatory cells (Figure 4). These newly formed blood vessels are more leaky than normal vessels and provide relatively easy access to the circulation for immediately adjacent tumour cells; for this reason, propensity to metastasize is directly related to the number of microvessels found in a growing tumour mass. Implications for therapy Recent attention has focused on the killing of endothelial cells in tumour-associated microvessels. This approach has theoretical advantages over conventional chemotherapy targeted at killing neoplastic cells. • The endothelial cells are unlikely to develop a classical drugresistance phenotype because they are normal cells. • Agents introduced into the circulation will have easy access to these endothelial cells. • As a consequence of the sluggish circulation within tumour vessels, the target endothelial cells will be exposed to cytotoxic agents for an extended period. • Destruction of a single tumour-associated blood vessel should destroy a core of tumour cells reliant on that vessel for metabolic viability (Figure 5). Recent experiments have demonstrated that natural inhibitors of tumour angiogenesis are produced by proteolytic cleavage of specific molecules in the body (e.g. plasminogen, collagen XVIII). Isolation and purification of these products (angiostatin and endostatin) was followed by the finding that their administration to animals with tumours resulted in rapid reduction of a range of large and fast-growing cancers. This reduction in gross tumour size
Angiogenesis (neovascularization) Unregulated proliferation at the individual cell level is commonly considered to be of central importance in neoplastic development. The tumour mass cannot grow beyond about 2 mm3 without establishing a new blood supply, however, because passive diffusion of nutrients and waste products is insufficient for the tumour’s metabolic needs. Efficient transfer of metabolites requires tumour cells to be within 200 µm of a capillary vessel, and this requires new vessels to expanding tumour foci. Neovascularization involves growth of columns of endothelial cells from pre-existing venules or capillaries towards the de-
Angiogenesis
Field effect of anti-angiogenic therapy 200 µm Angiogenic peptides
Destroying relatively few endothelial cells (red) in a small vessel supplying a core of tumour cells leads to the death of all the neoplastic cells within 200 µm of the blood vessel (blue). The neoplastic cells form a core around the patent lumen of this blood vessel and rely on it for their metabolic requirements.
New cords of endothelial cells are attracted to the tumour mass by angiogenic peptides. Anastomosis of these loops and formation of a patent lumen result in the establishment of a new nutritive supply, and also provide the cancer cells with easy access to the circulation. 5
4
MEDICINE
3
© 2004 The Medicine Publishing Company Ltd
CANCER BIOLOGY
modulation and that immunotherapy may affect tumour progression (as distinct from local tumour growth).
and decrease in growth rate was shown to result from inhibition of the growth of tumour vasculature. Presumably blocking of the angiogenic peptides that stimulate this new vessel development could also be a means of targeting the cancer. This appears to be the mechanism by which bevacizumab (a humanized antibody to vascular endothelial growth factor) shows promise as a first-line therapy for metastatic colorectal cancer.
Implications for therapy Several approaches to immunotherapy of tumours are based on the fact that various receptors regulating the immune response are expressed at the cell surface. For example, it is possible that cytotoxic T cells do not recognize tumours in the absence of major histocompatibility complex (MHC) class I antigens, and restoring or causing de novo expression of these molecules at the tumour cell surface (by biomodulation or gene therapy) might enable this process. Malignant melanoma is a common target for immunotherapy. High-dose interferon is under investigation as a possible means of stimulating the immune system against advanced melanoma; this intervention may act through induction of MHC class 1 antigens on tumour cells. CD8+ T cell response may be stimulated or restored by direct administration of various cytokines other than interferon. None of these approaches has been particularly successful, but research using dendritic cells in melanoma therapy appears promising. Dendritic cells are specific leucocytes that present antigens to T cells. It has recently been demonstrated that vaccination with autologous dendritic cells (pulsed with tumour lysates or with peptides representing known tumour antigens) produced objective responses in 30% of patients with advanced disease.
Loss of cell–cell cohesion in metastatic spread Growth of leaky blood vessels into a tumour deposit facilitates dissemination of cancer cells into the circulation. However, before cancer cells are released into the circulation, they must escape the cohesive constraints of the primary tumour mass. Cohesion is mediated by active homotypic (like-binds-like) cell adhesion molecules (CAMs). The most important CAMs belong to the cadherin family of cell surface receptors. These cadherins are transmembrane glycoproteins with a calcium-dependent ability to mediate cellular aggregation; though the different cadherins exhibit structural similarities, they tend to be expressed in a tissue-specific manner. Cadherin down-regulation results in loss of tissue integrity and is responsible for the breakdown of tissue architecture that permits the escape of individual cells. Abrogation of epithelial cadherin (E-cadherin) activity in carcinomas may occur as a consequence of down-regulation at the mRNA level, or may result from specific mutations in the E-cadherin gene (e.g. in lobular carcinoma of the breast). The role of cadherins in tumour development and progression was thought to relate solely to the effects of these molecules on tumour cell cohesion, but this is now considered a simplistic view. Down-regulation of a specific cadherin that modulates endothelial cell–cell interactions often occurs as a consequence of the action of cytokines released by neoplastic cells. This process, which is likely to occur during neovascularization, may exacerbate the naturally leaky nature of tumour blood vessels, thereby facilitating escape of neoplastic cells into the circulation.
Tumour cell arrest Disseminating cancer cells must lodge in the capillary beds of distant organs. This process may be passive (large emboli are simply blocked and mechanically trapped in smaller-diameter capillaries) or active. The trafficking of metastatic cancer cells resembles leucocyte extravasation at inflammatory sites, and the molecules involved in this normal phenomenon are also involved in cancer. Consequently, adhesion receptors (which are involved in regulating movement of WBCs out of the circulation at sites of inflammation) may be important in determining the behaviour of disseminating cancer cells. CD44, which was originally identified as a lymphocyte homing receptor, has various isoforms associated with the metastatic capacity of tumour cells, raising the possibil-
Implications for therapy It may be difficult to develop treatment based on cell cohesion research. Maintenance of epithelial integrity by up-regulation of E-cadherin activity (in the primary tumour mass or the endothelial lining of the blood vasculature) to prevent tumour spread would be desirable, but no agents are known to exert such an effect. Furthermore, almost 50% of patients with solid cancers have metastases at the time of presentation.
Attachment of tumour cells to activated endothelium a
Immune cell–tumour cell interactions Debate continues about the role of the immune system in modulating the growth of human tumours. Cloning and sequencing of tumour antigens has proved that these are expressed by human tumours, but, given that the primary tumour mass has managed to grow, the immune system does not appear to respond effectively to them. Small cancer cellular embolic aggregates (or even individual cells) are most likely to be exposed to host immune effector cells during dissemination through the circulation, and this is when maximal antitumour effects may occur. Many investigators believe that the process of metastasis is amenable to immunological
MEDICINE
b
c
Disseminating cancer (melanoma) cells produce cytokines (a) which up-regulate vascular cell adhesion molecule expression (b) on endothelial cells. The α4β1 integrin heterodimer expressed by circulating tumour cells mediates the binding interaction (c) with this ligand 6
4
© 2004 The Medicine Publishing Company Ltd
CANCER BIOLOGY
cilengitide (which preferentially targets αvβ3 integrin receptors) is being evaluated in phase II trials.
ity that anti-CD44 antibodies may preferentially target disseminating cancer cells. Specific receptor–ligand interactions on the endothelial cells lining the small capillaries and counter receptor– ligand interactions on the tumour cells might account for these general adhesive interactions during tumour cell arrest and for organ-specific patterns of metastasis. Targeting these interactions may not destroy the tumour cells directly, but could keep neoplastic cells continually within the hostile environment of the circulation. The adhesion receptors most responsible for tumour cell arrest probably belong to the integrin family of adhesive glycoproteins. These receptors primarily recognize constituents of the extracellular matrix and are involved in the migration of invading cancer cells through surrounding tissue. The integrins are transmembrane, heterodimeric glycoproteins formed from non-covalently associated α and β chains. The nature of the α and β constituents is responsible for the ligand specificity of these molecules and determines to which substrates the cells bind; for example, α5β1 is the classical fibronectin receptor, and cells expressing this heterodimer can attach to fibronectin. Integrins can also be involved in cell–cell interactions, with ligands that are members of the immunoglobulin superfamily. α4β1 is involved in cell–substrate interactions and also in cell–cell interactions; in the latter, the ligand is the vascular CAM (V-CAM), which is expressed on the surface of activated endothelium. α4β1 is expressed by WBCs and mediates binding of activated leucocytes to activated endothelium (i.e. with enhanced expression of specific ligands at the endothelial cell surface) at inflammatory sites, but it may also be involved in the binding of tumour cells to activated endothelium; α4β1 expression has been detected in the late stages of certain tumours (e.g. malignant melanoma). The fact that cancer cells can produce and secrete various cytokines capable of inducing the activated endothelial phenotype may explain how melanoma cancer cell arrest may occur (Figure 6). Thus, the α4β1 receptor recognizes and binds to V-CAM, expression of which has been up-regulated by cytokines secreted from the disseminating melanoma cell.
Tissue proteolysis The degradation of surrounding tissue barriers that accompanies tumour interactions with the extracellular matrix during invasion and extravasation is mediated by the production of degradative enzymes. These enzymes may be produced by cancer cells or may be induced as a result of stimulation by the cancer cells of surrounding stromal cells. The best-characterized proteolytic enzymes are the serine proteases and the matrix metalloproteinases. Between them, these enzymes can degrade all constituents of the extracellular matrix and provide the permissive environment into which actively migrating tumour cells can move. Because unrestricted protein degradation would be disadvantageous to the organism, both tumour cells and the normal cells surrounding them produce specific inhibitors of these proteases – the tissue inhibitors of metalloproteinases and plasminogen activator inhibitors 1 and 2, which block the activity and degradative capacity of these enzymes. Implications for therapy There have been numerous attempts to develop synthetic inhibitors of protease activity. In recent clinical studies, these agents have been used in ovarian, gastric and pancreatic cancer. Generally, the results of these antiproteolytic treatments have been disappointing, and this might suggest that targeting the enzymatic activity of disseminating tumour cells is too late an intervention to show real therapeutic promise. However, the parallel between the invasive capacity of malignant tumour cells and the invasive behaviour of migrating endothelial cells during tumour angiogenesis means that any observed efficacy might result from antineovascularization rather than a directly inhibitory effect on tumour cell invasion.
Implications for therapy Attempts are being made to prevent the release of inflammatory cytokines from neoplastic cells, to control the level of expression of the α4β1 heterodimer, and to inhibit V-CAM-1 up-regulation in experimental models. The rationale behind these approaches is that, by reducing cell adherence and maintaining disseminating cancer cells within the hostile environment of the circulation, cancer cells may die, thus preventing tumour spread. If it was known exactly when tumour cells were to be shed into the circulation, anti-adhesive therapy might be feasible – otherwise, timing of delivery is a major practical difficulty. Some anti-angiogenic therapies under consideration depend on anti-integrin approaches. αvβ3 (the so-called vitronectin receptor) is expressed only on the surface of dividing, not quiescent, endothelial cells. Monoclonal antibodies that recognize the αvβ3 heterodimer or arginine–glycine–aspartic acid peptides (the recognition sequence for many integrins) have been shown to have anti-angiogenic activities in targeting dividing endothelial cells (causing them to undergo apoptosis) and thereby affecting tumour growth. Anti-angiogenesis clinical trials based on these treatments are now under way in the USA, and the cyclic Arg–Gly–Asp peptide
MEDICINE
FURTHER READING Franks L M, Teich N M, eds. Introduction to the cellular and molecular biology of cancer. 3rd ed. Oxford: Oxford University Press, 1997.
5
© 2004 The Medicine Publishing Company Ltd
What’s new ?
Biology of cancer
• Gene therapy approaches to the treatment of cancer have been developed, based on the expression of wild-type versions of mutated tumour-suppressor genes
Ian R Hart
• Several specific tyrosine kinase inhibitors have been introduced into clinical practice • The first successful trial of anti-angiogenic treatment has been reported, in metastatic colorectal cancer Cancer is a spectrum of genetic diseases resulting from various mutations in specific genes. These mutations, which may either abrogate gene function or increase gene function (by causing constitutive activation), drive cells toward the unregulated and irreversible cellular proliferation that is the hallmark of cancer. Over the last 10–15 years, considerable progress has been made in defining and understanding these changes at the molecular level. Development of novel therapeutic approaches derived from this increased understanding has been slower, but it is hoped that elucidation of the biochemical pathways involved in tumour development, progression and metastasis will identify novel targets against which agents can be developed to regulate these processes. Some are already entering clinical trials. This contribution focuses on the areas of cancer biology that appear to offer the greatest opportunity for short-term development of novel therapeutic agents, and shows how increased understanding of the pathogenesis of malignancy can provide a means of developing such treatments.
lular DNA of animals, where they did not operate as cancer genes. It appears that the viral (v-onc) genes were acquired by viruses from normal cells during viral evolution and were integrated into the virus. Subsequently, these genes caused tumorigenicity when expressed in virus-infected cells. Tumorigenicity could result from
Experiment showing that DNA contains genes capable of conferring a tumorigenic phenotype on putatively normal cells a High molecular weight tumour DNA precipitated with calcium phosphate
b Normal recipient cells (murine 3T3 fibroblasts)
Development of cancer Dominant-acting oncogenes Epidemiological studies have shown that about 80% of cancers result from environmental factors. These factors lead to gradual acquisition of a series of mutations in target cells, resulting in the progression of a normal phenotype to a neoplastic one. These mutations often confer a survival benefit on the cells, leading to preferential growth of a new focus of mutant cells that can become a target for further mutations. In the early 1980s, a series of elegant experiments involving transfer of only tumour-derived DNA to recipient cells demonstrated that the transformed phenotype could be acquired in a dominant manner (Figure 1). It became apparent that many of the DNA sequences causing these changes in recipient cell transformation status were already known – they had been identified as oncogenes (onc), which were additional sequences within the viral genomes of RNA tumour viruses (retroviruses). These onc genes were shown to be almost identical to sequences in the normal cel-
c Focus formation
d Foci grow as tumours in mice
High molecular weight DNA isolated from tumour is precipitated with calcium phosphate and transfected into normal, non-tumorigenic recipient cells of the murine 3T3 fibroblast line. In the presence of transforming DNA, the 3T3 cells pile up and form evident foci in the Petri dish. Inoculation of such cells into recipient mice leads to the formation of rapidly growing fibrosarcomas. Alternatively, when these transformed cells are plated in soft agar they grow in an anchorage-independent manner.
Ian R Hart is Professor of Tumour Biology at Barts and The London, Queen Mary’s School of Medicine and Dentistry, London, UK. He qualified as a veterinary surgeon from the University of Bristol, and undertook research at the University of Bristol and in the USA. His research interests include the role of cell adhesion in tumour spread.
MEDICINE
e Anchorage independent in soft agar
1
1
© 2004 The Medicine Publishing Company Ltd
CANCER BIOLOGY
over-expression of the encoded product, or from differences in sequence between the viral and cellular homologues – the transforming DNA identified by transfection techniques (using DNA isolated from cancers) contained comparable alterations. These oncogenes appear to actively cause increased cell proliferation. They are thought to lie within the biochemical pathways driving cellular proliferation, and could: • be members of the tyrosine kinase family (responsible for phosphorylating downstream proteins) • have GTPase activity (i.e. bind guanosine triphosphate and convert it to the diphosphate and monophosphate forms), which is involved in signal transduction cascades (transmission of signals from cell surface to nucleus) • be related to growth factors or growth factor receptors • regulate gene expression by binding to DNA, as a result of their nuclear localization.
coli gene, p53, and the breast cancer 1 (BRCA1) and 2 (BRCA2) genes. In normal organisms, cell proliferation is balanced by regulated cell loss, often through apoptosis (programmed cell death). Many tumour-suppressor genes are involved in cell proliferation, in genetic stability and in cell death, and they are often mutated in cancer; such mutations in these genes generally ablate the ability to cause apoptosis and result in increased cell numbers. Implications for therapy Understanding of the biochemical pathways that drive cellular proliferation may lead to development of specific inhibitors; for example, tyrosine kinase inhibitors may block phosphorylation of proteins involved in the signalling that induces cell proliferation, whereas inhibitors of GTPase could prevent activity of oncogenes through this pathway. Alternatively, if mutated tumour-suppressor genes can be replaced, cellular proliferation can be suppressed and apoptosis in the target cell population may increase. This concept underlies gene therapy protocols aimed at re-expressing tumoursuppressor genes (e.g. p53) specifically in tumour cells. Recent reports of an international phase II/III trial of p53 gene delivery as a first-line treatment in ovarian cancer were disappointing, but in such gene-therapy protocols the major limiting factor is almost certainly the efficiency of gene delivery. The best example of a novel approach to cancer therapy based on the role of oncogenes in tumour biology is the use of a monoclonal antibody that recognizes the HER2-neu (c-erbB2) oncogene. HER2-neu encodes a transmembrane tyrosine kinase receptor with extensive homology to the epidermal growth factor
Tumour-suppressor genes Tumour-suppressor genes were first suggested in studies of rare inherited cancer syndromes. In familial retinoblastoma, for example, a genetic change was inherited through the germ line, but mutation or ablation of the other allele on the homologous chromosome was required for tumour development (Figure 2). In sporadic cases, independent mutational events in both homologous genes are required; as a result, sporadic retinoblastoma is less common than the familial form. Many tumour-suppressor genes have now been identified, including the retinoblastoma gene, the adenomatous polyposis
Inherited
The role of herceptin in preventing HER2/neu dimerization and subsequent generation of a signal transduction event
Sporadic
HER2/neu receptor Rwt
Rdel
Rwt
Ligand binding
Blockade
Rwt
Step 1
Herceptin Cell membrane
m
R
R
del
wt
R
R
m
P
P
Step 2 P P
Rm
Rm
P P
Inactive receptor monomer is activated by ligand binding, which brings together the monomers to form active dimers. Dimerization results in transient activation and phosphorylation of specific tyrosine residues within the intracellular domain. These phosphorylation events initiate signals that result in changes in gene expression and are associated with growth of the tumour. The herceptin antibody blocks binding of the ligand to the receptor.
In sporadic cancers such as retinoblastoma, two mutations are required to eliminate the retinoblastoma gene. In inherited cases, a mutated or deleted copy is inherited in the germ line and thus only one event is needed in any cell to eliminate both copies of the gene. 2
MEDICINE
P P
3
2
© 2004 The Medicine Publishing Company Ltd
CANCER BIOLOGY
receptor (Figure 3). Ligands that bind to the extracellular domain of this receptor induce dimerization and internalization, and activate intracellular tyrosine kinase activity. Gene amplification of HER2neu has been documented in up to one-third of breast cancers, and a similar proportion have increased protein levels in the absence of gene amplification. Herceptin is an anti-HER2 antibody that has been tested in a phase III clinical trial. Alternatively, rather than attempting to block the extracellular receptor, it might be possible to develop small molecules that inhibit the downstream sequelae of binding of the appropriate ligand to the receptor. This approach has been used with several tyrosine kinase inhibitors including ZD1839, which is proposed to inhibit signalling from epidermal growth factor receptors. The most successful agent of this type is perhaps ST1-571 (imatinib), which efficiently inhibits the Bcr-Abl oncoprotein. This tumour-specific tyrosine kinase is produced through fusion of the ABL and BCR genes, which occurs as a result of chromosomal translocations (the Philadelphia chromosome) in chronic myeloid leukaemia.
veloping tumour mass. New vessels are induced to form as a result of the release of a variety of angiogenic peptides, which may be produced by the neoplastic cells or by host inflammatory cells (Figure 4). These newly formed blood vessels are more leaky than normal vessels and provide relatively easy access to the circulation for immediately adjacent tumour cells; for this reason, propensity to metastasize is directly related to the number of microvessels found in a growing tumour mass. Implications for therapy Recent attention has focused on the killing of endothelial cells in tumour-associated microvessels. This approach has theoretical advantages over conventional chemotherapy targeted at killing neoplastic cells. • The endothelial cells are unlikely to develop a classical drugresistance phenotype because they are normal cells. • Agents introduced into the circulation will have easy access to these endothelial cells. • As a consequence of the sluggish circulation within tumour vessels, the target endothelial cells will be exposed to cytotoxic agents for an extended period. • Destruction of a single tumour-associated blood vessel should destroy a core of tumour cells reliant on that vessel for metabolic viability (Figure 5). Recent experiments have demonstrated that natural inhibitors of tumour angiogenesis are produced by proteolytic cleavage of specific molecules in the body (e.g. plasminogen, collagen XVIII). Isolation and purification of these products (angiostatin and endostatin) was followed by the finding that their administration to animals with tumours resulted in rapid reduction of a range of large and fast-growing cancers. This reduction in gross tumour size
Angiogenesis (neovascularization) Unregulated proliferation at the individual cell level is commonly considered to be of central importance in neoplastic development. The tumour mass cannot grow beyond about 2 mm3 without establishing a new blood supply, however, because passive diffusion of nutrients and waste products is insufficient for the tumour’s metabolic needs. Efficient transfer of metabolites requires tumour cells to be within 200 µm of a capillary vessel, and this requires new vessels to expanding tumour foci. Neovascularization involves growth of columns of endothelial cells from pre-existing venules or capillaries towards the de-
Angiogenesis
Field effect of anti-angiogenic therapy 200 µm Angiogenic peptides
Destroying relatively few endothelial cells (red) in a small vessel supplying a core of tumour cells leads to the death of all the neoplastic cells within 200 µm of the blood vessel (blue). The neoplastic cells form a core around the patent lumen of this blood vessel and rely on it for their metabolic requirements.
New cords of endothelial cells are attracted to the tumour mass by angiogenic peptides. Anastomosis of these loops and formation of a patent lumen result in the establishment of a new nutritive supply, and also provide the cancer cells with easy access to the circulation. 5
4
MEDICINE
3
© 2004 The Medicine Publishing Company Ltd
CANCER BIOLOGY
modulation and that immunotherapy may affect tumour progression (as distinct from local tumour growth).
and decrease in growth rate was shown to result from inhibition of the growth of tumour vasculature. Presumably blocking of the angiogenic peptides that stimulate this new vessel development could also be a means of targeting the cancer. This appears to be the mechanism by which bevacizumab (a humanized antibody to vascular endothelial growth factor) shows promise as a first-line therapy for metastatic colorectal cancer.
Implications for therapy Several approaches to immunotherapy of tumours are based on the fact that various receptors regulating the immune response are expressed at the cell surface. For example, it is possible that cytotoxic T cells do not recognize tumours in the absence of major histocompatibility complex (MHC) class I antigens, and restoring or causing de novo expression of these molecules at the tumour cell surface (by biomodulation or gene therapy) might enable this process. Malignant melanoma is a common target for immunotherapy. High-dose interferon is under investigation as a possible means of stimulating the immune system against advanced melanoma; this intervention may act through induction of MHC class 1 antigens on tumour cells. CD8+ T cell response may be stimulated or restored by direct administration of various cytokines other than interferon. None of these approaches has been particularly successful, but research using dendritic cells in melanoma therapy appears promising. Dendritic cells are specific leucocytes that present antigens to T cells. It has recently been demonstrated that vaccination with autologous dendritic cells (pulsed with tumour lysates or with peptides representing known tumour antigens) produced objective responses in 30% of patients with advanced disease.
Loss of cell–cell cohesion in metastatic spread Growth of leaky blood vessels into a tumour deposit facilitates dissemination of cancer cells into the circulation. However, before cancer cells are released into the circulation, they must escape the cohesive constraints of the primary tumour mass. Cohesion is mediated by active homotypic (like-binds-like) cell adhesion molecules (CAMs). The most important CAMs belong to the cadherin family of cell surface receptors. These cadherins are transmembrane glycoproteins with a calcium-dependent ability to mediate cellular aggregation; though the different cadherins exhibit structural similarities, they tend to be expressed in a tissue-specific manner. Cadherin down-regulation results in loss of tissue integrity and is responsible for the breakdown of tissue architecture that permits the escape of individual cells. Abrogation of epithelial cadherin (E-cadherin) activity in carcinomas may occur as a consequence of down-regulation at the mRNA level, or may result from specific mutations in the E-cadherin gene (e.g. in lobular carcinoma of the breast). The role of cadherins in tumour development and progression was thought to relate solely to the effects of these molecules on tumour cell cohesion, but this is now considered a simplistic view. Down-regulation of a specific cadherin that modulates endothelial cell–cell interactions often occurs as a consequence of the action of cytokines released by neoplastic cells. This process, which is likely to occur during neovascularization, may exacerbate the naturally leaky nature of tumour blood vessels, thereby facilitating escape of neoplastic cells into the circulation.
Tumour cell arrest Disseminating cancer cells must lodge in the capillary beds of distant organs. This process may be passive (large emboli are simply blocked and mechanically trapped in smaller-diameter capillaries) or active. The trafficking of metastatic cancer cells resembles leucocyte extravasation at inflammatory sites, and the molecules involved in this normal phenomenon are also involved in cancer. Consequently, adhesion receptors (which are involved in regulating movement of WBCs out of the circulation at sites of inflammation) may be important in determining the behaviour of disseminating cancer cells. CD44, which was originally identified as a lymphocyte homing receptor, has various isoforms associated with the metastatic capacity of tumour cells, raising the possibil-
Implications for therapy It may be difficult to develop treatment based on cell cohesion research. Maintenance of epithelial integrity by up-regulation of E-cadherin activity (in the primary tumour mass or the endothelial lining of the blood vasculature) to prevent tumour spread would be desirable, but no agents are known to exert such an effect. Furthermore, almost 50% of patients with solid cancers have metastases at the time of presentation.
Attachment of tumour cells to activated endothelium a
Immune cell–tumour cell interactions Debate continues about the role of the immune system in modulating the growth of human tumours. Cloning and sequencing of tumour antigens has proved that these are expressed by human tumours, but, given that the primary tumour mass has managed to grow, the immune system does not appear to respond effectively to them. Small cancer cellular embolic aggregates (or even individual cells) are most likely to be exposed to host immune effector cells during dissemination through the circulation, and this is when maximal antitumour effects may occur. Many investigators believe that the process of metastasis is amenable to immunological
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b
c
Disseminating cancer (melanoma) cells produce cytokines (a) which up-regulate vascular cell adhesion molecule expression (b) on endothelial cells. The α4β1 integrin heterodimer expressed by circulating tumour cells mediates the binding interaction (c) with this ligand 6
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CANCER BIOLOGY
cilengitide (which preferentially targets αvβ3 integrin receptors) is being evaluated in phase II trials.
ity that anti-CD44 antibodies may preferentially target disseminating cancer cells. Specific receptor–ligand interactions on the endothelial cells lining the small capillaries and counter receptor– ligand interactions on the tumour cells might account for these general adhesive interactions during tumour cell arrest and for organ-specific patterns of metastasis. Targeting these interactions may not destroy the tumour cells directly, but could keep neoplastic cells continually within the hostile environment of the circulation. The adhesion receptors most responsible for tumour cell arrest probably belong to the integrin family of adhesive glycoproteins. These receptors primarily recognize constituents of the extracellular matrix and are involved in the migration of invading cancer cells through surrounding tissue. The integrins are transmembrane, heterodimeric glycoproteins formed from non-covalently associated α and β chains. The nature of the α and β constituents is responsible for the ligand specificity of these molecules and determines to which substrates the cells bind; for example, α5β1 is the classical fibronectin receptor, and cells expressing this heterodimer can attach to fibronectin. Integrins can also be involved in cell–cell interactions, with ligands that are members of the immunoglobulin superfamily. α4β1 is involved in cell–substrate interactions and also in cell–cell interactions; in the latter, the ligand is the vascular CAM (V-CAM), which is expressed on the surface of activated endothelium. α4β1 is expressed by WBCs and mediates binding of activated leucocytes to activated endothelium (i.e. with enhanced expression of specific ligands at the endothelial cell surface) at inflammatory sites, but it may also be involved in the binding of tumour cells to activated endothelium; α4β1 expression has been detected in the late stages of certain tumours (e.g. malignant melanoma). The fact that cancer cells can produce and secrete various cytokines capable of inducing the activated endothelial phenotype may explain how melanoma cancer cell arrest may occur (Figure 6). Thus, the α4β1 receptor recognizes and binds to V-CAM, expression of which has been up-regulated by cytokines secreted from the disseminating melanoma cell.
Tissue proteolysis The degradation of surrounding tissue barriers that accompanies tumour interactions with the extracellular matrix during invasion and extravasation is mediated by the production of degradative enzymes. These enzymes may be produced by cancer cells or may be induced as a result of stimulation by the cancer cells of surrounding stromal cells. The best-characterized proteolytic enzymes are the serine proteases and the matrix metalloproteinases. Between them, these enzymes can degrade all constituents of the extracellular matrix and provide the permissive environment into which actively migrating tumour cells can move. Because unrestricted protein degradation would be disadvantageous to the organism, both tumour cells and the normal cells surrounding them produce specific inhibitors of these proteases – the tissue inhibitors of metalloproteinases and plasminogen activator inhibitors 1 and 2, which block the activity and degradative capacity of these enzymes. Implications for therapy There have been numerous attempts to develop synthetic inhibitors of protease activity. In recent clinical studies, these agents have been used in ovarian, gastric and pancreatic cancer. Generally, the results of these antiproteolytic treatments have been disappointing, and this might suggest that targeting the enzymatic activity of disseminating tumour cells is too late an intervention to show real therapeutic promise. However, the parallel between the invasive capacity of malignant tumour cells and the invasive behaviour of migrating endothelial cells during tumour angiogenesis means that any observed efficacy might result from antineovascularization rather than a directly inhibitory effect on tumour cell invasion.
Implications for therapy Attempts are being made to prevent the release of inflammatory cytokines from neoplastic cells, to control the level of expression of the α4β1 heterodimer, and to inhibit V-CAM-1 up-regulation in experimental models. The rationale behind these approaches is that, by reducing cell adherence and maintaining disseminating cancer cells within the hostile environment of the circulation, cancer cells may die, thus preventing tumour spread. If it was known exactly when tumour cells were to be shed into the circulation, anti-adhesive therapy might be feasible – otherwise, timing of delivery is a major practical difficulty. Some anti-angiogenic therapies under consideration depend on anti-integrin approaches. αvβ3 (the so-called vitronectin receptor) is expressed only on the surface of dividing, not quiescent, endothelial cells. Monoclonal antibodies that recognize the αvβ3 heterodimer or arginine–glycine–aspartic acid peptides (the recognition sequence for many integrins) have been shown to have anti-angiogenic activities in targeting dividing endothelial cells (causing them to undergo apoptosis) and thereby affecting tumour growth. Anti-angiogenesis clinical trials based on these treatments are now under way in the USA, and the cyclic Arg–Gly–Asp peptide
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FURTHER READING Franks L M, Teich N M, eds. Introduction to the cellular and molecular biology of cancer. 3rd ed. Oxford: Oxford University Press, 1997.
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