hall 17_01
Tissue homeostasis depends on the regulated cell division and self-elimination (programmed cell death) of each of its constituent members except its stem cells. In fact, a tumor arises as a result of uncontrolled cell division and failure for self-elimination. One can consider cancer as a Darwinian-like process whereby the fittest cells reproduce to become the dominant population of a tumor. Alterations in three groups of genes are responsible for the deregulated control mechanisms that are the hallmarks of cancer cells:
1. Proto-oncogenes are components of signaling networks that act as positive growth regulators in response to mitogens, cytokines, and cell-to- cell contact. A gain-of-function mutation in only one copy of a proto-oncogene results in a dominantly acting oncogene that often fails to respond to extracellular signals.
2. Tumor-suppressor genes are also components of the same signaling networks as proto-oncogenes, except that they act as negative growth regulators. They modulate proliferation and survival by antagonizing the biochemical functions of protooncogenes or responding to unchecked growth signals. In contrast to oncogenes, inactivation of both copies of tumor-suppressor genes is required for loss of function in most cases.
3. DNA stability genes form a class of genes involved in both monitoring and maintaining the integrity of DNA. Loss of these genes results in defective sensing of DNA lesions as well as improper repair of the damaged template.
The malignant progression from normal tissue to tumor to metastasis occurs in a number of discrete “steps” over a period of time. These steps, which are the result of mutations, deletions, or gene changes in the three groups of genes described here, may occur spontaneously as a consequence of random errors or result from exposure to agents as diverse as chemical mutagens, ionizing radiations, ultraviolet light, and viruses and provide a growth or survival advantage that allows the cells to become the clonal origin of the tumor. To summarize, tumor evolution results from the accumulation of gene mutations that arise in a single cell that has suffered a disruption in its regulatory mechanisms for proliferation, self-elimination, immortalization, and genetic stability. This is illustrated in Figure 17.1.
MECHANISMS OF CARCINOGENESIS
A single genetic alteration that leads to the activation of an oncogene or loss of a tumor-suppressor gene does not by itself lead to the formation of a solid tumor. Instead, carcinogenesis appears to be a multistep process with multiple genetic alterations occurring over an extended period of time; at least, that is how it appears. Sometimes these genetic alterations are carried in the germ line, as, for example, in the cancer-predisposing syndrome retinoblastoma; however, heritable mutations are rare. Most alterations that lead to cancer are acquired in the form of somatic mutations: chromosomal translocations, deletions, inversions, amplifications, or simple point mutations.
Initially, it was thought that cancer was the result of deregulated growth signals by oncogenes, a concept supported by increased proliferation in many types of cancer. In the last decade, the finding that many cancers possess diminished apoptotic (programmed cell death) programs or loss of cell- cycle control has led to the concept that mutations in proto-oncogenes and tumor-suppressor genes that inhibit apoptosis provide a selective growth advantage to a premalignant cell that allows it to clonally expand. Mutations in DNA stability genes increase the rate of acquiring genetic mutations that will result in a malignant tumor. Thus, while tumor cells are considered clonal in origin, most tumors contain heterogeneous populations of cells that differ in their ability to repopulate the tumor or form metastasis. In fact, only a small percentage of tumor cells possess the ability to form a tumor, leading to the concept that tumors possess “stem cells” and that elimination of these stem cells is essential for controlling tumor growth.
ONCOGENES
The first demonstration that a tumor was initiated by a cellular component found in tumor cells but not normal cells was shown by Rous in the early 1900s. His landmark studies demonstrated that cell-free extracts derived from chicken sarcomas could cause a new sarcoma if injected into healthy chickens. In the l970s, with the advent of molecular biology, several groups identified the etiological agent for sarcoma formation in chickens as an RNA virus, designated the Rous sarcoma virus (RSV), which belongs to a group of viruses designated retroviruses - viruses whose genomes are of RNA. Thus, oncogenes were first discovered from a study of retroviruses that cause cancers in animals. Although the virus had been identified, it still remained to be elucidated how this retrovirus causes a sarcoma, since another virus belonging to this same group of RNA viruses, avian leukosis virus (ALV), docs not transform cells in culture or induce sarcomas. Analysis of the genomes of ALV and RSV revealed that RSV contains approximately 1,500 more base pairs of DNA than ALV. It was hypothesized, therefore, that these extra base pairs of DNA in the RSV genome are responsible for the tumorigenic activity. This was supported by the observation that deletion mutants of RSV that are missing this 1,500-hp region lose their transformation potential but can still replicate and produce viral progeny normally. This led to the conclusion that the transforming activity and replicative activity of RSV are encoded by genetically distinct regions of the virus and that only a small portion of the RSV genome is needed for transformation.
From these early studies, several important conclusions could be derived: (1) Cancer can be caused by a genetically transmissible agent - in the case of chicken sarcoma, by a retrovirus containing a unique piece of genetic information that was latter designated the src gene; (2) only a certain region of a retrovirus is needed for transformation; and (3) the region of the viral genome necessary for transformation is not involved in the normal replicative life cycle.
Huebner and Todaro later proposed that cancer- causing viral genes such as src are normally inactive but can be activated when they recombine with a retroviral genome. Once they do so, they pass from being a benign proto-oncogene (i.e., c-crc) to a malignant form (v-src) capable of causing cancer when introduced into the appropriate host cell. Although we now know that viruses represent only one of several mechanisms that cause the deregulated expression of a proto-oncogene, these studies helped to define oncogenes as mutant forms of normal cellular genes that are altered in expression and/or function by various agents, including radiation, chemicals, and viruses. Consequently, very different agents produce tumors that are indistinguishable one from another. This is illustrated in Figure 17.2.
MECHANISMS OF ONCOGENE ACTIVATION
Although many mechanisms are involved in oncogene activation, transcriptional deregulation by overexpression or abnormal expression of the mRNA of a proto-oncogene is a common theme. At least four mechanisms exist for oncogene activation in human neoplasms (Fig.
17.3).
Retroviral Integration through Recombination
Retroviral integration of proto-oncogene sequences in retroviral genomes occurs through two possible recombinational mechanisms. In the first, mRNAs from a proto-oncogene recombine with viral genomic RNAs. During the recombination process, the proto-oncogene mRNA becomes deregulated as it comes under the control of the viral promoter, termed LTR. However, the probability of RNA recombination events between proto-oncogene mRNA and viral mRNA generating an oncogenic retrovirus is quite low and undermines the importance of this mechanism.
A second more probable mechanism is as follows: First a retroviral genome integrates in dose proximity to a proto-oncogene, where the proto-oncogene is under the transcriptional control of the retrovirus LTR promoter. Then the viral and proto-oncogene sequences become closely associated through a DNA recombination event that permits the production of mRNAs that contain both viral and proto-oncogene sequences. In this scenario, the proto-oncogene becomes transcriptionally deregulated as it is under the control of the viral promoter LTR. In addition. it can acquire mutations in its coding sequence. Although the proto-oncogene can become mutated during the recombination process, the key point is that its deregulated expression by the viral LTR increases its expression and promotes cell growth.
DNA Mutation of Regulatory Sites
The union of the technique for gene transfer with mouse transformation assays facilitated the isolation of human oncogenes that were activated by DNA mutation. Transfection of human DNA into immortalized but untransformed mouse cells was first used to isolate the H-ras oncogene from bladder carcinoma cells. The key to this approach is that only transformed cells possess the ability to grow in soft agar (Fig. 17.4). The implicit assumption is that a specifìc gene (or more than one) is responsible for causing the bladder carcinoma and that it will act in a dominant fashion to induce a tumor. Indeed, multiple groups were successful in isolating the H-ras oncogene by this approach.
Several steps are needed for the molecular cloning of an oncogene from transformed rodent cells using the rodent fibroblast transformation assay. First, human DNA containing the transforming oncogene is transfected into mouse cells, and then DNA from the transformed mouse cells is serially transfected to reduce the amount of human DNA that is not associated with the transforming oncogene. After several rounds of transfection, the DNA is isolated from a soft agar colony and digested with restriction enzymes to make a genomic DNA library. The library is then screened with a human-specific repetitive probe that does not cross-react with mouse DNA, thereby identifying human sequences in a mouse background. Clones that possess human repetitive sequences are then isolated and digested with restriction enzymes to identify a similar-length fragment that is common to all transformants. Finally, DNA from the clones is transfected into mouse cells to confirm its oncogenic potential. If the oncogene is present in this genomic clone, then a significant percentage of the transfected mouse cells should be transformed when compared with transfecting genomic DNA from untransformed cells.
Perhaps the prototypical example of oncogene activation by DNA mutation is the H-ras oncogene. The H-ras oncogene was isolated by the approach just described, and its DNA sequence was compared with its normal cellular counterpart. At first comparison, there did not seem to be any difference between oncogenic and proto-oncogenic forms. However, because H-ras is a relatively small oncogene. It was possible to sequence the entire gene to rigorously search for small mutations. It did not take long to find the difference between the two forms of the gene. The transforming, oncogenic H-ras gene possesses a single base-pair mutation that changes the 12th amino acid from glycine to valine. This single DNA mutation is responsible for changing H-ras from a benign proto-oncogene into a malìgnant oncogene. We now know that mutations in codons 13 and 61 will also produce oncogenic H-ras genes that are constitutively locked in an active state.
Gene Amplification
Oncogene amplification occurs through bridge breakage fusion cycles in anaphase during mitosis. In contrast to the other mechanisms discussed that involve transcriptional deregulation as a key mechanism of oncogene activation, gene amplification represents an alternative means of increasing proto-oncogene expression by increasing the number of DNA copies of the proto-oncogene. Gene amplification can result in an increased number of copies of extrachromosomal molecules called double minutes or can result in intrachromosomal amplified regions called homogeneously staining regions (HSRs), both of which are detectable by fluorescence in situ hybridization or Giemsa banding of chromosomes. The N-myc oncogene is a classic example of an oncogene amplified in leukemia, neuroblastoma, and breast cancer.
Chromosome Translocation
It had long been known that tumors possessed abnormal karyotypes. However, the chromosome content of many solid tumors is unstable, making it difficult to determine which cytogenetic alterations are causative for tumorigenesis and which are the consequence of the neoplastic process. The first real breakthrough in identifying tumor-specific chromosome alterations occurred in the late 1950s when Dr. Peter Nowell found a consistent shortened version of chromosome 22 in individuals afflicted with chronic myelogenous leukemia (CML). Because many patients with CML possess an abnormal chromosome 22 in their leukemic cells, this was a strong indication that a specific chromosome alteration is involved in the pathogenesis of this malignancy. With the advent of more sophisticated cytogenetic and molecular techniques, it was discovered that this shortened version of chromosome 22 is due to a symmetric translocation with chromosome 9. It was hypothesized. therefore, that the translocation between chromosomes 9 and 22 gives rise to CML. Further molecular analysis revealed that the bcr gene on chromosome 9 translocates in front of the abl gene on chromosome 22, producing a fusion transcript with abnormal expression (Fig. 17.5).
With the recent advent of molecular cytogenetics - that is, fluorescent in situ hybridization (FTSH) - many translocation partners have been identified. In fact, a common strategy has been to use proto-oncogenes, which chromosomally map near translocation breakpoints, as markers to identify potential translocation partners. Although numerous translocation breakpoints have been identified in hematopoietic neoplasms, few consistent translocations have been found in solid-tissue tumors. The reason for this is still unclear, but may be attributed to the fact that hematopoietic cancers require fewer alterations for neoplasia than solid tumors. Table 17.1 provides examples of the chromosomal changes that result in oncogene activation and the associated human malignancies. Interestingly, there are no known examples of oncogenes activated by retroviruses in human malignancies.
MUTATION AND INACTIVATION OF TUMOR-SUPPRESSOR GENES
The Retinoblastoma Paradigm
Oncogenes result from a mutation, deletion, or alteration in the expression of one copy of a gene. Thus, oncogenes are dominant genes, because a mutation in only one copy will cause their activation, even though the other copy of the gene is unchanged. This concept led to speculation that another class of genes, termed “anti-oncogenes,” suppress the effect of oncogenes on transformation and tumor formation. The existence of tumor suppressor genes was supported by cell fusion studies between tumor ceils and normal cells and by the family history studies of people afflicted with inherited cancer-prone disorders such as retinoblastoma or Li - Fraumeni syndrome. Although one mutated version of an oncogene is sufficient to drive malignant progression, one functional copy of a tumor suppressor gene is sufficient to suppress transformation, suggesting that both copies of a tumor suppressor gene must be inactivated to inhibit tumor growth (Fig. 17.6). Insight into the mechanism of tumor-suppressor gene inactivation came from Knudson’s epidemiological studies of families in which retinoblastoma appeared to be inherited in an autosomal dominant manner. Patients with familial retinoblastoma develop bilateral or multifocal disease at an earlier age than patients with sporadic retinoblastoma. Based on these observations, Knudson proposed that in the inherited form of retinoblastoma, individuals possess a gem-line mutation of the retinoblastoma gene (Rb) in all the cells of their body, but inactivation of one Rb allele does not give rise to retinoblastoma. Thus, the disease appeared to be autosomal dominant because individuals are born with one mutated allele. However, a second mutation in a retinal cell is required to develop retinoblastoma. In the sporadic form of the disease, an individual has to acquire two Rb mutations in the same retinal cell to develop retinoblastoma. The “two-hit hypothesis” by Knudson provided a genetic basis to understand the differences in inherited and sporadic mutations in the onset of tumors and to advance the concept that both alleles of a tumor-suppressor gene need to he inactivated to promote tumor development. Thus, tumor-suppressor genes are recessive genes that require the inactivation of both functional gene copies before malignancies develop, whereas loss of one functional copy results only in increased cancer susceptibility. In addition, it is often the case that the same tumor-suppressor gene involved in hereditary cancer syndromes, such as retinoblastoma, is also inactivated in other forms of cancer. The Rb gene itself has now been implicated in several other human cancers, which indicates that it may play a
generalized role in tumor growth suppression in a variety of tissues. For example, patients who are cured of familial retinoblastoma are at increased risk of osteosarcoma, small-cell lung cancer, and breast cancer; although the loss of the Rh gene alone is sufficient for retinoblastoma, further changes are required for the development of these other tumors.
The Li - Fraumeni Paradigm
The Li - Fraumeni syndrome (LPS) is a rare autosomal dominantly inherited disease that predisposes individuals to develop osteosarcomas, soft tissue sarcomas, rhabdomyosarcomas, leukemias, brain tumors, and carcinomas of the lung and breast (Fig. 17.7). Initial attempts to identify the genetic mutations that underlie LPS were unsuccessful because of the rarity of the syndrome and the high mortality of the patients. The major insight into the underlying cause of Li - Fraumeni came when it was found that mice that overexpress a mutant version of the p53 tumor-suppressor gene in the presence of the wild-type pS3 gene develop a spectrum of tumors similar to that seen in Li - Fraumeni patients. Sequencing of p53 in affected family members revealed germ-line missense mutations in the p53 tumor-suppressor gene located on chromosome 17p13 that resulted in its inactivation, and tumors derived from affected individuals had lost the remaining wild-type allele of p53. Similar to retinoblastoma, loss or inactivation of both wild type copies of p53 is needed for tumor formation. However, functional loss of one germ-line inherited copy of mutant p53 accelerates the onset of spontaneous tumor formation. Therefore, Li - Fraumeni patients follow a similar paradigm as retinoblastoma patients in developing spontaneous tumors, but unlike retinoblastoma, in which germ-line mutations mainly give rise to retinal tumors, loss of p53 results in a wide spectrum of tumors. Table 17.2 provides examples of other cancer predisposition genes and their associated syndromes.
SOMATIC HOMOZYGOSITY
How are recessive tumor-suppressor genes lost? Cytogenetic studies are used to identify chromosomal changes in peripheral blood lymphocytes or fibroblasts from cancer patients, especially those with a family history of cancer, to identify chromosomal rearrangements or deletions. At the subchromosomal level, genome-wide linkage analysis is used to determine that a certain chromosome region is tightly linked with cancer predisposition. Both copies of a suppressor gene in the sporadic form of retinoblastoma and other solid tumors may result from two independent allelic mutations, but in practice, it occurs more often by the process of somatic homozygosity. The steps appear to be as follows: One chromosome of a pair is lost, a deletion occurs in the remaining chromosome, and the chromosome with the deletion replicates. Instead of having each of the two alleles contributed by different parents, the cell has both alleles from the same parent, with loss of a vital piece containing the tumor-suppressor gene (Fig. 17.8). This process has been documented for chromosome 13 in the case of retinoblastoma, chromosome 11 in Wilms’ tumor, chromosome 3 for small-cell lung cancer, and chromosome 5 for colon cancer. Most interesting of all, perhaps, is the case of astrocytomas, in which somatic homozygosity is observed for chromosome 10 in grade 11 and III astrocytoma and for both chromosomes 10 and 17 for grade IV glioblastoma.
THE MULTISTEP NATURE OF CANCER
Perhaps the most pervasive dogma in cancer research is that carcinogenesis is a multistage process. The implication is that there are a number of distinct events that may be separated in time. This idea is almost 70 years old and is exemplified by the skin cancer experiments in mice that introduced the concepts of initiation, promotion, and progression as stages in tumor development.
Genetic analysis of cells from solid tumors, too, suggests alterations, mutations, or deletions in multiple signaling genes, either oncogenes or suppressor genes; 6 to 12 mutations have been suggested for the formation of a carcinoma. In the case of colorectal cancer, a model has been proposed that correlates a series of chromosomal and molecular events with the changes in the histopathology of normal epithelium during the multistage formation of colorectal cancer and metastatic carcinoma. This concept is illustrated in Figure 17.9.
A more general model of the series of events in carcinogenesis is shown in Figure 17.10. The first event, from whatever cause (including ionizing radiation), causes a mutation in a gene in one of the families responsible for the stability of the genome. This leads to a mutator phenotype, so that with many cells dividing, multiple mutations are likely in cancer-associated genes, both oncogenes and tumor-suppressor genes. This in turn leads to progression of the cancer and ultimately its invasive and metastatic properties.
Therefore, it is not surprising that restoration of one copy of a tumor-suppressor gene is sometimes not sufficient to restore tumor-suppressor activity, as solid tumors accumulate mutations that can make them refractory to the restoration of a single tumor suppressor gene. Although tumor-suppressor genes are functionally quite different from oncogenes, they modulate similar cellular targets as oncogenes.
1. Proto-oncogenes are components of signaling networks that act as positive growth regulators in response to mitogens, cytokines, and cell-to- cell contact. A gain-of-function mutation in only one copy of a proto-oncogene results in a dominantly acting oncogene that often fails to respond to extracellular signals.
2. Tumor-suppressor genes are also components of the same signaling networks as proto-oncogenes, except that they act as negative growth regulators. They modulate proliferation and survival by antagonizing the biochemical functions of protooncogenes or responding to unchecked growth signals. In contrast to oncogenes, inactivation of both copies of tumor-suppressor genes is required for loss of function in most cases.
3. DNA stability genes form a class of genes involved in both monitoring and maintaining the integrity of DNA. Loss of these genes results in defective sensing of DNA lesions as well as improper repair of the damaged template.
The malignant progression from normal tissue to tumor to metastasis occurs in a number of discrete “steps” over a period of time. These steps, which are the result of mutations, deletions, or gene changes in the three groups of genes described here, may occur spontaneously as a consequence of random errors or result from exposure to agents as diverse as chemical mutagens, ionizing radiations, ultraviolet light, and viruses and provide a growth or survival advantage that allows the cells to become the clonal origin of the tumor. To summarize, tumor evolution results from the accumulation of gene mutations that arise in a single cell that has suffered a disruption in its regulatory mechanisms for proliferation, self-elimination, immortalization, and genetic stability. This is illustrated in Figure 17.1.
MECHANISMS OF CARCINOGENESIS
A single genetic alteration that leads to the activation of an oncogene or loss of a tumor-suppressor gene does not by itself lead to the formation of a solid tumor. Instead, carcinogenesis appears to be a multistep process with multiple genetic alterations occurring over an extended period of time; at least, that is how it appears. Sometimes these genetic alterations are carried in the germ line, as, for example, in the cancer-predisposing syndrome retinoblastoma; however, heritable mutations are rare. Most alterations that lead to cancer are acquired in the form of somatic mutations: chromosomal translocations, deletions, inversions, amplifications, or simple point mutations.
Initially, it was thought that cancer was the result of deregulated growth signals by oncogenes, a concept supported by increased proliferation in many types of cancer. In the last decade, the finding that many cancers possess diminished apoptotic (programmed cell death) programs or loss of cell- cycle control has led to the concept that mutations in proto-oncogenes and tumor-suppressor genes that inhibit apoptosis provide a selective growth advantage to a premalignant cell that allows it to clonally expand. Mutations in DNA stability genes increase the rate of acquiring genetic mutations that will result in a malignant tumor. Thus, while tumor cells are considered clonal in origin, most tumors contain heterogeneous populations of cells that differ in their ability to repopulate the tumor or form metastasis. In fact, only a small percentage of tumor cells possess the ability to form a tumor, leading to the concept that tumors possess “stem cells” and that elimination of these stem cells is essential for controlling tumor growth.
ONCOGENES
The first demonstration that a tumor was initiated by a cellular component found in tumor cells but not normal cells was shown by Rous in the early 1900s. His landmark studies demonstrated that cell-free extracts derived from chicken sarcomas could cause a new sarcoma if injected into healthy chickens. In the l970s, with the advent of molecular biology, several groups identified the etiological agent for sarcoma formation in chickens as an RNA virus, designated the Rous sarcoma virus (RSV), which belongs to a group of viruses designated retroviruses - viruses whose genomes are of RNA. Thus, oncogenes were first discovered from a study of retroviruses that cause cancers in animals. Although the virus had been identified, it still remained to be elucidated how this retrovirus causes a sarcoma, since another virus belonging to this same group of RNA viruses, avian leukosis virus (ALV), docs not transform cells in culture or induce sarcomas. Analysis of the genomes of ALV and RSV revealed that RSV contains approximately 1,500 more base pairs of DNA than ALV. It was hypothesized, therefore, that these extra base pairs of DNA in the RSV genome are responsible for the tumorigenic activity. This was supported by the observation that deletion mutants of RSV that are missing this 1,500-hp region lose their transformation potential but can still replicate and produce viral progeny normally. This led to the conclusion that the transforming activity and replicative activity of RSV are encoded by genetically distinct regions of the virus and that only a small portion of the RSV genome is needed for transformation.
From these early studies, several important conclusions could be derived: (1) Cancer can be caused by a genetically transmissible agent - in the case of chicken sarcoma, by a retrovirus containing a unique piece of genetic information that was latter designated the src gene; (2) only a certain region of a retrovirus is needed for transformation; and (3) the region of the viral genome necessary for transformation is not involved in the normal replicative life cycle.
Huebner and Todaro later proposed that cancer- causing viral genes such as src are normally inactive but can be activated when they recombine with a retroviral genome. Once they do so, they pass from being a benign proto-oncogene (i.e., c-crc) to a malignant form (v-src) capable of causing cancer when introduced into the appropriate host cell. Although we now know that viruses represent only one of several mechanisms that cause the deregulated expression of a proto-oncogene, these studies helped to define oncogenes as mutant forms of normal cellular genes that are altered in expression and/or function by various agents, including radiation, chemicals, and viruses. Consequently, very different agents produce tumors that are indistinguishable one from another. This is illustrated in Figure 17.2.
MECHANISMS OF ONCOGENE ACTIVATION
Although many mechanisms are involved in oncogene activation, transcriptional deregulation by overexpression or abnormal expression of the mRNA of a proto-oncogene is a common theme. At least four mechanisms exist for oncogene activation in human neoplasms (Fig.
17.3).
Retroviral Integration through Recombination
Retroviral integration of proto-oncogene sequences in retroviral genomes occurs through two possible recombinational mechanisms. In the first, mRNAs from a proto-oncogene recombine with viral genomic RNAs. During the recombination process, the proto-oncogene mRNA becomes deregulated as it comes under the control of the viral promoter, termed LTR. However, the probability of RNA recombination events between proto-oncogene mRNA and viral mRNA generating an oncogenic retrovirus is quite low and undermines the importance of this mechanism.
A second more probable mechanism is as follows: First a retroviral genome integrates in dose proximity to a proto-oncogene, where the proto-oncogene is under the transcriptional control of the retrovirus LTR promoter. Then the viral and proto-oncogene sequences become closely associated through a DNA recombination event that permits the production of mRNAs that contain both viral and proto-oncogene sequences. In this scenario, the proto-oncogene becomes transcriptionally deregulated as it is under the control of the viral promoter LTR. In addition. it can acquire mutations in its coding sequence. Although the proto-oncogene can become mutated during the recombination process, the key point is that its deregulated expression by the viral LTR increases its expression and promotes cell growth.
DNA Mutation of Regulatory Sites
The union of the technique for gene transfer with mouse transformation assays facilitated the isolation of human oncogenes that were activated by DNA mutation. Transfection of human DNA into immortalized but untransformed mouse cells was first used to isolate the H-ras oncogene from bladder carcinoma cells. The key to this approach is that only transformed cells possess the ability to grow in soft agar (Fig. 17.4). The implicit assumption is that a specifìc gene (or more than one) is responsible for causing the bladder carcinoma and that it will act in a dominant fashion to induce a tumor. Indeed, multiple groups were successful in isolating the H-ras oncogene by this approach.
Several steps are needed for the molecular cloning of an oncogene from transformed rodent cells using the rodent fibroblast transformation assay. First, human DNA containing the transforming oncogene is transfected into mouse cells, and then DNA from the transformed mouse cells is serially transfected to reduce the amount of human DNA that is not associated with the transforming oncogene. After several rounds of transfection, the DNA is isolated from a soft agar colony and digested with restriction enzymes to make a genomic DNA library. The library is then screened with a human-specific repetitive probe that does not cross-react with mouse DNA, thereby identifying human sequences in a mouse background. Clones that possess human repetitive sequences are then isolated and digested with restriction enzymes to identify a similar-length fragment that is common to all transformants. Finally, DNA from the clones is transfected into mouse cells to confirm its oncogenic potential. If the oncogene is present in this genomic clone, then a significant percentage of the transfected mouse cells should be transformed when compared with transfecting genomic DNA from untransformed cells.
Perhaps the prototypical example of oncogene activation by DNA mutation is the H-ras oncogene. The H-ras oncogene was isolated by the approach just described, and its DNA sequence was compared with its normal cellular counterpart. At first comparison, there did not seem to be any difference between oncogenic and proto-oncogenic forms. However, because H-ras is a relatively small oncogene. It was possible to sequence the entire gene to rigorously search for small mutations. It did not take long to find the difference between the two forms of the gene. The transforming, oncogenic H-ras gene possesses a single base-pair mutation that changes the 12th amino acid from glycine to valine. This single DNA mutation is responsible for changing H-ras from a benign proto-oncogene into a malìgnant oncogene. We now know that mutations in codons 13 and 61 will also produce oncogenic H-ras genes that are constitutively locked in an active state.
Gene Amplification
Oncogene amplification occurs through bridge breakage fusion cycles in anaphase during mitosis. In contrast to the other mechanisms discussed that involve transcriptional deregulation as a key mechanism of oncogene activation, gene amplification represents an alternative means of increasing proto-oncogene expression by increasing the number of DNA copies of the proto-oncogene. Gene amplification can result in an increased number of copies of extrachromosomal molecules called double minutes or can result in intrachromosomal amplified regions called homogeneously staining regions (HSRs), both of which are detectable by fluorescence in situ hybridization or Giemsa banding of chromosomes. The N-myc oncogene is a classic example of an oncogene amplified in leukemia, neuroblastoma, and breast cancer.
Chromosome Translocation
It had long been known that tumors possessed abnormal karyotypes. However, the chromosome content of many solid tumors is unstable, making it difficult to determine which cytogenetic alterations are causative for tumorigenesis and which are the consequence of the neoplastic process. The first real breakthrough in identifying tumor-specific chromosome alterations occurred in the late 1950s when Dr. Peter Nowell found a consistent shortened version of chromosome 22 in individuals afflicted with chronic myelogenous leukemia (CML). Because many patients with CML possess an abnormal chromosome 22 in their leukemic cells, this was a strong indication that a specific chromosome alteration is involved in the pathogenesis of this malignancy. With the advent of more sophisticated cytogenetic and molecular techniques, it was discovered that this shortened version of chromosome 22 is due to a symmetric translocation with chromosome 9. It was hypothesized. therefore, that the translocation between chromosomes 9 and 22 gives rise to CML. Further molecular analysis revealed that the bcr gene on chromosome 9 translocates in front of the abl gene on chromosome 22, producing a fusion transcript with abnormal expression (Fig. 17.5).
With the recent advent of molecular cytogenetics - that is, fluorescent in situ hybridization (FTSH) - many translocation partners have been identified. In fact, a common strategy has been to use proto-oncogenes, which chromosomally map near translocation breakpoints, as markers to identify potential translocation partners. Although numerous translocation breakpoints have been identified in hematopoietic neoplasms, few consistent translocations have been found in solid-tissue tumors. The reason for this is still unclear, but may be attributed to the fact that hematopoietic cancers require fewer alterations for neoplasia than solid tumors. Table 17.1 provides examples of the chromosomal changes that result in oncogene activation and the associated human malignancies. Interestingly, there are no known examples of oncogenes activated by retroviruses in human malignancies.
MUTATION AND INACTIVATION OF TUMOR-SUPPRESSOR GENES
The Retinoblastoma Paradigm
Oncogenes result from a mutation, deletion, or alteration in the expression of one copy of a gene. Thus, oncogenes are dominant genes, because a mutation in only one copy will cause their activation, even though the other copy of the gene is unchanged. This concept led to speculation that another class of genes, termed “anti-oncogenes,” suppress the effect of oncogenes on transformation and tumor formation. The existence of tumor suppressor genes was supported by cell fusion studies between tumor ceils and normal cells and by the family history studies of people afflicted with inherited cancer-prone disorders such as retinoblastoma or Li - Fraumeni syndrome. Although one mutated version of an oncogene is sufficient to drive malignant progression, one functional copy of a tumor suppressor gene is sufficient to suppress transformation, suggesting that both copies of a tumor suppressor gene must be inactivated to inhibit tumor growth (Fig. 17.6). Insight into the mechanism of tumor-suppressor gene inactivation came from Knudson’s epidemiological studies of families in which retinoblastoma appeared to be inherited in an autosomal dominant manner. Patients with familial retinoblastoma develop bilateral or multifocal disease at an earlier age than patients with sporadic retinoblastoma. Based on these observations, Knudson proposed that in the inherited form of retinoblastoma, individuals possess a gem-line mutation of the retinoblastoma gene (Rb) in all the cells of their body, but inactivation of one Rb allele does not give rise to retinoblastoma. Thus, the disease appeared to be autosomal dominant because individuals are born with one mutated allele. However, a second mutation in a retinal cell is required to develop retinoblastoma. In the sporadic form of the disease, an individual has to acquire two Rb mutations in the same retinal cell to develop retinoblastoma. The “two-hit hypothesis” by Knudson provided a genetic basis to understand the differences in inherited and sporadic mutations in the onset of tumors and to advance the concept that both alleles of a tumor-suppressor gene need to he inactivated to promote tumor development. Thus, tumor-suppressor genes are recessive genes that require the inactivation of both functional gene copies before malignancies develop, whereas loss of one functional copy results only in increased cancer susceptibility. In addition, it is often the case that the same tumor-suppressor gene involved in hereditary cancer syndromes, such as retinoblastoma, is also inactivated in other forms of cancer. The Rb gene itself has now been implicated in several other human cancers, which indicates that it may play a
generalized role in tumor growth suppression in a variety of tissues. For example, patients who are cured of familial retinoblastoma are at increased risk of osteosarcoma, small-cell lung cancer, and breast cancer; although the loss of the Rh gene alone is sufficient for retinoblastoma, further changes are required for the development of these other tumors.
The Li - Fraumeni Paradigm
The Li - Fraumeni syndrome (LPS) is a rare autosomal dominantly inherited disease that predisposes individuals to develop osteosarcomas, soft tissue sarcomas, rhabdomyosarcomas, leukemias, brain tumors, and carcinomas of the lung and breast (Fig. 17.7). Initial attempts to identify the genetic mutations that underlie LPS were unsuccessful because of the rarity of the syndrome and the high mortality of the patients. The major insight into the underlying cause of Li - Fraumeni came when it was found that mice that overexpress a mutant version of the p53 tumor-suppressor gene in the presence of the wild-type pS3 gene develop a spectrum of tumors similar to that seen in Li - Fraumeni patients. Sequencing of p53 in affected family members revealed germ-line missense mutations in the p53 tumor-suppressor gene located on chromosome 17p13 that resulted in its inactivation, and tumors derived from affected individuals had lost the remaining wild-type allele of p53. Similar to retinoblastoma, loss or inactivation of both wild type copies of p53 is needed for tumor formation. However, functional loss of one germ-line inherited copy of mutant p53 accelerates the onset of spontaneous tumor formation. Therefore, Li - Fraumeni patients follow a similar paradigm as retinoblastoma patients in developing spontaneous tumors, but unlike retinoblastoma, in which germ-line mutations mainly give rise to retinal tumors, loss of p53 results in a wide spectrum of tumors. Table 17.2 provides examples of other cancer predisposition genes and their associated syndromes.
SOMATIC HOMOZYGOSITY
How are recessive tumor-suppressor genes lost? Cytogenetic studies are used to identify chromosomal changes in peripheral blood lymphocytes or fibroblasts from cancer patients, especially those with a family history of cancer, to identify chromosomal rearrangements or deletions. At the subchromosomal level, genome-wide linkage analysis is used to determine that a certain chromosome region is tightly linked with cancer predisposition. Both copies of a suppressor gene in the sporadic form of retinoblastoma and other solid tumors may result from two independent allelic mutations, but in practice, it occurs more often by the process of somatic homozygosity. The steps appear to be as follows: One chromosome of a pair is lost, a deletion occurs in the remaining chromosome, and the chromosome with the deletion replicates. Instead of having each of the two alleles contributed by different parents, the cell has both alleles from the same parent, with loss of a vital piece containing the tumor-suppressor gene (Fig. 17.8). This process has been documented for chromosome 13 in the case of retinoblastoma, chromosome 11 in Wilms’ tumor, chromosome 3 for small-cell lung cancer, and chromosome 5 for colon cancer. Most interesting of all, perhaps, is the case of astrocytomas, in which somatic homozygosity is observed for chromosome 10 in grade 11 and III astrocytoma and for both chromosomes 10 and 17 for grade IV glioblastoma.
THE MULTISTEP NATURE OF CANCER
Perhaps the most pervasive dogma in cancer research is that carcinogenesis is a multistage process. The implication is that there are a number of distinct events that may be separated in time. This idea is almost 70 years old and is exemplified by the skin cancer experiments in mice that introduced the concepts of initiation, promotion, and progression as stages in tumor development.
Genetic analysis of cells from solid tumors, too, suggests alterations, mutations, or deletions in multiple signaling genes, either oncogenes or suppressor genes; 6 to 12 mutations have been suggested for the formation of a carcinoma. In the case of colorectal cancer, a model has been proposed that correlates a series of chromosomal and molecular events with the changes in the histopathology of normal epithelium during the multistage formation of colorectal cancer and metastatic carcinoma. This concept is illustrated in Figure 17.9.
A more general model of the series of events in carcinogenesis is shown in Figure 17.10. The first event, from whatever cause (including ionizing radiation), causes a mutation in a gene in one of the families responsible for the stability of the genome. This leads to a mutator phenotype, so that with many cells dividing, multiple mutations are likely in cancer-associated genes, both oncogenes and tumor-suppressor genes. This in turn leads to progression of the cancer and ultimately its invasive and metastatic properties.
Therefore, it is not surprising that restoration of one copy of a tumor-suppressor gene is sometimes not sufficient to restore tumor-suppressor activity, as solid tumors accumulate mutations that can make them refractory to the restoration of a single tumor suppressor gene. Although tumor-suppressor genes are functionally quite different from oncogenes, they modulate similar cellular targets as oncogenes.
posted by stefanoxx at 1:07 AM
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