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Etiology of Osteosarcoma

Fuchs, Bruno*,**; Pritchard, Douglas, J.*

Clinical Orthopaedics and Related Research: April 2002 - Volume 397 - Issue - p 40-52
SECTION I SYMPOSIUM: Papers Presented at the 2000 and 2001 Meetings of the Musculoskeletal Tumor Society: Tumor Biology
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Although the prognosis and quality of life of patients with osteosarcoma were improved significantly during the past decades, the pathogenesis and etiology of this disease remain obscure. Significant interest and effort in this cancer led to the identification of numerous etiologic agents. Several chemical agents such as beryllium, viruses such as FBJ, subsequently found to contain the src-oncogene, and radiation were shown to be potent inducers of osteosarcoma. Paget’s disease, electrical burn, or trauma all are thought to be other factors that may contribute to the pathogenesis. More recently, patients with hereditary diseases such as Rothmund-Thomson syndrome, Bloom syndrome, and Li-Fraumeni syndrome were found to have an increased risk of having osteosarcoma develop. During the past few years, the molecular analysis brought a wealth of new information with numerous genes that were associated with osteosarcoma and its clinical disease progression. They can be categorized into self-sufficiency in growth signals, insensitivity to growth inhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue evasion and metastasis. Although the understanding of these processes in osteosarcoma still is incomplete, it may have the potential to significantly affect the patient care in the future.

From the Mayo Clinic, *Department of Orthopedics and **Department of Biochemistry & Molecular Biology, Tumor Biology Program, Rochester, MN.

B.F. was supported by a scholarship from the Schweizerische Gesellschaft für Orthopädie SGO.

Reprint requests to D.J. Pritchard, MD, Division of Orthopedic Oncology, Department of Orthopedics, Mayo Clinic, 200 First Street SW, Rochester, MN 55905.

List of Abbreviations Used: BMP bone morphogenetic protein, DNA deoxyribonucleic acid, ECM extracellular matrix, FBJ virus naturally occurring murine sarcoma virus, first described by Finkel, Biskis, and Jinkins, FGF fibroblast growth factor, HER2 (erbB-2) human EGF receptor, IGF insulinlike growth factor, mRNA messenger ribonucleic acid, PDGF platelet-derived growth factor, RNA ribonucleic acid, TGF tumor growth factor, TNF tumor necrosis factor, VEGF vascular endothelial growth factor

Osteosarcoma is of interest to every orthopaedic surgeon. Although the incidence of osteosarcoma is much lower than for many other forms of cancer, there are certain aspects that intrigue. Why does osteosarcoma tend to occur in adolescents when most other cancers occur in older people? Why does osteosarcoma tend to occur in the distal femur or proximal tibia? Why does osteosarcoma seem to arise in areas of preexisting Paget’s disease? There are numerous unanswered questions about this cancer, one of which regards the etiology. The precise cause of osteosarcoma is unknown but there are some hints that may be helpful in eventually unraveling this intriguing question. Recently, great effort has been spent particularly on delineating the molecular aspects of carcinogenesis. Even though there is a wealth of new information, there is an obligation to apply these new insights in the clinical practice for the benefit of the patients. It becomes evident that the development of a tumor, an osteosarcoma in particular, is based on a complicated network of different events at the genetic level. The current overview focuses on current general aspects of carcinogenesis and tries to highlight, besides known risk factors, the actual understanding of osteosarcoma at the molecular level. However, during the past decades, many diverse initiating factors in conjunction with the development of osteosarcoma have been identified. It is not clear how such initiating agents and the genetic events are related.

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Etiologic Agents

Known etiologic agents or initiating factors may be categorized into chemical agents, viruses, radiation, and other miscellaneous.

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Chemical Agents

Numerous chemicals are known to induce osteosarcoma, including methylcholanthrene, 7,107 beryllium oxide, 20 and zinc beryllium silicate. 5,14,35,46,95 Gardner and Heslington 35 produced osteosarcoma in rabbits by the intravenous injection of various beryllium compounds and observed splenic atrophy was associated with the development of osteosarcoma. Janes et al 46 worked with zinc beryllium silicate in rabbits and observed that splenic atrophy occurred only in rabbits that had osteosarcoma develop. All rabbits that previously had splenectomy had osteosarcoma develop, whereas only approximately ½ of the rabbits that did not have splenectomy had osteosarcoma develop.

In 1961, the previous supply of zinc beryllium silicate became unavailable and a new source failed to produce osteosarcoma. It was thought that this substance had a lower concentration of free beryllium oxide. 83

Peterson and coworkers 79 studied the ultrastructure of beryllium-induced osteosarcoma in the rabbit and found no evidence of intracellular particles of the zinc beryllium silicate. Presumably the beryllium acts as an initiator or trigger, perhaps leading to a genetic alteration. This experience with beryllium suggested that there may be a role for trace metals in carcinogenesis.

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Radiation

One of the earliest known mechanisms of osteosarcoma induction involved the radium dial painters in the 1920s. Many of these workers, mostly women, would lick the ends of their brushes to get a fine point as they painted radium on the watch dials to make them glow in the dark. 9,27 Some of these women had osteosarcoma develop. By the time of the Manhattan project to develop an atomic bomb in World War II, knowledge of the consequences of exposure to radium suggested caution in the use of radioactive materials. 83

New studies were instituted with internal and external radiation in laboratory animals. One of the major research efforts was the determination of the biologic toxicity of plutonium, heavy metals, and the fission products, which tend to localize in bone. Therefore, bone cancer, as a late manifestation, became an area of major interest to those working with radioactive materials. 26,28–30,65,82

It was well known that radiation exposure was related to the development of osteosarcoma. Biologic studies were initiated at Argonne National Laboratory when the Manhattan project moved from the University of Chicago. 25 Some of these studies involved an attempt at establishing a minimum radiation dose, using a large number of beagles exposed to continuous radiation and followed up for many years. Various tumors resulted including osteosarcoma. Much technical information regarding accumulated radiation dosage and the rate at which the dosage was delivered has been published. 26,28 Numerous investigators have studied the oncogenic effects of radiation. 29,30,66,82 It is known that patients who receive radiation as treatment for one disease may have osteosarcoma develop within the radiation field.

This especially is true for patients with Ewing’s sarcoma who receive radiation as part of their treatment. A concominent delivery of anthracycline chemotherapy significantly shortens the interval between radiation treatment and the development of osteosarcoma. It now is known that radiation can cause osteosarcoma regardless of the source of the radiation; the minimum dose still is not established, although it is known that it takes a certain minimum period between exposure to radiation and the development of cancer. From the practical standpoint, this usually is said to be at least 4 years in humans. 83

Thorotrast is a patented radioactive agent, which was injected as a contrast medium for imaging studies and formerly was widely used. 44 It has been associated with the development of bone sarcomas and other cancers, especially liver, after a long latency. This agent no longer is used, but there are some patients who received this agent who still are at risk.

Approximately all radionuclides that localize in bone can produce bone cancer in the laboratory. Young, growing bone retains more bone-seeking radionuclides than does adult bone. Other factors that are important are the size and type of bone. Large bones are more responsive to radionuclides with strong beta particles than are small bones because the proportion of available energy absorbed increased as bone size increases.

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Viruses

The relationship of viruses to the development of sarcomas is interesting. Rous et al 88 found that when a cell-free extract from chicken fibrosarcomas was injected into other chicks, they developed new sarcomas. These studies began in 1911. The transponding agent subsequently was known to be a virus called the Rous sarcoma virus. This proved to be a retrovirus whose RNA genome is reverse transcribed into DNA, which is incorporated into the host cell genome. The Rous sarcoma virus contains a gene called v-Src. V-Src has been identified as an oncogene. It subsequently has been learned that normal cells from chickens and some other species contain a related normal gene called c-Src, a protooncogene. 83 Therefore, there is a relationship between a viral oncogene and a normal cellular protooncogene suggesting that cancer may be induced by the action of normal or nearly normal genes.

Oncogenic viruses are thought to have arisen by transducing a normal cellular protooncogene into their genome, a subsequent mutation in the transduced gene then converts it into an oncogene. 29 Many oncogenes derived from cellular protooncogenes have been identified in different retroviruses, implying that the normal vertebrate genome contains many potential cancer causing genes.

Additional viral studies were done at the Argonne National Laboratory as an off-shoot of the biologic studies, which already were underway regarding radiation effects. 28,30

A virus called FBJ was isolated from a spontaneously occurring sarcoma in a mouse and was shown to be an extremely potent inducer of osteosarcoma. 27 Oncogenic viruses in general are classified as containing either RNA or DNA. The C-type RNA viruses usually induce leukemia, lymphoma, or sarcomas in various species, whereas the B-type RNA virus induces murine mammary carcinomas. Oncogenic DNA viruses usually produce only solid carcinomas and sarcomas. 38 The DNA viruses known to produce bone tumors include polyoma virus 93 and Simian Virus-40. 19 Of the many viruses that cause tumors in certain experimental animals, only the FBJ mouse virus, as a C-type particle, has been isolated from a spontaneously occurring sarcoma.

Osteosarcomas induced by FBJ virus show numerous C-type particles on electron microscopy. The virus is unique because it consistently produces tumors only in bone (except for those tumors that arise in the inoculated area).

FBJ is a very potent virus, even when diluted many thousand times, when inoculated into newborn mice; approximately 100% of the mice have osteosarcoma develop. Often multiple bony metastases occur, but lung metastases are relatively uncommon in the mouse.

It is of some interest that there is another virus which induces benign bone tumors in mice, which is called RFB virus, again a C-type of virus. 30

When this benign virus is injected into newborn mice on Day 1 and FBJ virus is injected on Day 2, none of the mice had malignant bone tumors develop. When the reverse is tried, that is, the FBJ is given on Day 1 and the benign virus is inoculated on Day 2, approximately all of the mice had osteosarcoma develop. The reason for this is not understood entirely.

Additional experience at the Argonne National Laboratory also was of interest. Cell-free extracts were isolated from osteosarcomas from humans and inoculated into Syrian hamsters. 19 A significant number of hamsters injected with these cell-free extracts of human bone cancer had different tumors develop including 20 osteosarcomas and 11 fibrosarcomas, whereas only one tumor, a fibrosarcoma, developed in the control animals. These investigators also did some interaction studies with bone seeking radionuclides and the bone tumor-inducing viruses that led to the hypothesis that radiation produces cancer by inactivating a viral inhibitor or a tumor suppressor. 29

One of the most interesting spin-offs of the FBJ virus studies is the understanding that the viral gene present in FBJ is identical to the v-Fos gene, which is important in the modern molecular biology theory of cancer induction. v-Fos is an oncogene. The corresponding natural gene found in the cell is called c-Fos and is considered to be a protooncogene. v-Fos enters a cell nucleus, attaches to DNA, and alters it. If v-Fos contains a mutated gene, this will affect the cell’s function, which perhaps will lead to unregulated bone formation.

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Other Miscellaneous Causes

Numerous other associations related to the development of osteosarcoma have been described. Is trauma an initiating factor? 69 It is doubtful; however, there are cases such as a policeman who accidentally shot himself in the thigh and some years later had osteosarcoma develop at this site. Is this a coincidence? Is it related to the bullet fragments? It is not known.

Osteosarcoma has been found in association with an electrical burn. 1 It also has been found adjacent to a surgical sponge left in a dog. 77 Another investigator induced osteosarcoma by the use of subperiosteal sheathing with plastic film. 76 This remains one of the most puzzling forms of oncogenesis. Perhaps anoxia is induced leading to some sort of damage to DNA analogous to damage caused by radiation. 15 There even is a suspected relationship between the fluoridation of drinking water and osteosarcoma. There is one report of osteosarcoma found in association with total joint replacements and other metallic implants; a frightening prospect. 51 The relation of Paget’s disease and osteosarcoma is not well understood. Approximately 1% of patients with Paget’s disease have osteosarcoma develop. This constitutes an increase in risk that is several thousand-fold higher than the general population. Recent analyses, however, showed a genetic predisposition. Pagetic and sporadic osteosarcoma showed a loss of heterozygosity for all or part of the distal portion of chromosome 18. 42,67

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Hereditary Factors

There are numerous inherited syndromes in which osteosarcoma develops. Rothmund-Thomson syndrome is one of the syndromes and is characterized clinically by early onset of poikiloderma, short stature, sparseness of eyebrows, lashes, or both, juvenile cataracts, sunlight sensitivity, hypogonadism, defective dentition, and nail abnormalities. 60 Because the two clinical syndromes defined by Rothmund 87 and Thomson 96 were recognized as an entity, 11 more than 200 patients were reported in the literature. 100 Rothmund-Thomson syndrome is associated with an increased risk for cancer, such as cutaneous epitheliomas (basal, squamous, Morbus Bowen), gastric adenocarcinomas, fibrosarcomas, and osteosarcomas. 17 Since the first report of a patient with Rothmund-Thomson syndrome having an osteosarcoma develop, 86 more than 16 cases have been reported, 92 and the increased risk of these patients having osteosarcoma develop is accepted. 71 These rare but striking clinical findings initiated an intense cytogenetic and molecular analysis. 62 In a team effort gathering information or tissue samples from seven patients with Rothmund-Thomson syndrome, the genetic defect (GGCCTGC deletion or a C→T point mutation) recently could be targeted to the RECQL4 gene on chromosome 8q24.3. RECQL4 encodes a DNA helicase that unwinds double-stranded DNA into single-stranded DNA, which is, if altered, responsible for causing Rothmund-Thomson syndrome. 52 Bloom syndrome, first described as a congenital telangiectatic erythema resembling lupus erythematosus in patients with dwarfism, 6 is another inherited disorder in which the patients have an increased risk of having osteosarcoma develop. It is characterized clinically by prenatal and postnatal growth retardation, a thin triangular face, telangiectactic rash in sun exposed areas, birth weight less than 2500 g at term, normal intelligence, and its association to cancers. 105 As of 1996, 100 cancers were reported in 71 of 168 registered individuals referred to the Bloom syndrome registry. 36 The Bloom syndrome gene (BME) could be located on chromosome 15q26.1 and also encodes for a RECQ helicase. 21 Rothmund-Thomson and Bloom syndromes are two examples of how knowledge of a disease can be improved by in-depth study of rare diseases providing an understanding of fundamental biologic processes. A third inherited disorder for which the patient has a risk of having osteosarcoma develop is Li-Fraumeni syndrome. This syndrome is defined clinically by any proband younger than 45 years with a sarcoma having a first-degree relative younger than 45 years with any cancer and an additional first- or second-degree relative in the same lineage with any cancer or sarcoma at any age. 61 Patients with Li-Fraumeni syndrome are at high risk of having osteosarcoma and soft tissue sarcomas, breast cancers, brain tumors, acute leukemia, adrenal cortical cancers, and gonadal giant cell tumors. 84 The genetic defect lies in a mutation in the p53-suppressor gene on chromosome 17p13, exon 7. 65 The TP53 gene encodes the p53 tumor suppressor protein, which is responsible for monitoring the integrity of the genome, 57 and the G1/S-checkpoint control after DNA damage, 55 and it leads to cell cycle arrest, 49 apoptosis, 13 and DNA repair. 31 TP53 gene mutations, however, are detected only in 71% of families with classic Li-Fraumeni syndrome, implying other mechanisms causing this classic clinical entity. 98

Another hint about the etiology of osteosarcoma came from the association that was discovered between certain forms of retinoblastoma and the subsequent development of osteosarcoma. It initially was thought that these cases occurred because of radiation treatment to the region of the orbit, but it subsequently was learned that osteosarcomas could occur in any part of the body and not just in the region of the orbit. 58

Deoxyribonucleic acid samples from children with this tumor showed that there was an inherited mutation in the Rb gene. Retinoblastoma occurs in inheritable autosomal dominant form, which often is bilateral, and a sporadic form, which is later in onset and usually unilateral. Sequential deletions of both allelic genes in the Rb locus on chromosome 13–14 are required for the development of retinoblastoma in either form. This was explained by the two-hit model, the first mutation may be one that is inherited at birth in the germ line in the hereditable form or it may be acquired later by somatic mutation in the sporadic form. The second mutation, the second hit, at the same locus, is somatic and random in either form. 3,54 Both mutations are necessary for neoplastic transformation of a retinal cell to retinoblastoma. The germ line inheritance of a mutated Rb also predisposes to the development of osteosarcoma and other cancers. The normal Rb gene is a transcription regulator and functions in the regulation of the cell cycle and the suppression of tumor cell proferation. Loss of this function removes the regulatory control of the Rb. The Rb and the TP53 genes belonged to the first to be associated with the etiology of osteosarcoma. Meanwhile, a better understanding with respect to the molecular carcinogenesis in general has emerged, and numerous other genes and their alterations are implicated in the conversion of a normal cell into a malignant cell. 33

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Molecular Etiology of Osteosarcoma

Malignancy is a state that emerges from a tumor-host microenvironment in which the host participates in the induction, selection, and expansion of the neoplastic process. 63 The evolutionary nature of cancer offers numerous important insights into the malignant process Strikingly, the development of a cancer cell is a rare event. With an estimated mutation rate of some 1 in 2 × 107 per gene cell division targeting cells in the average human, cancer occurs in only one in three lifetimes. 74 Moreover, only the rare and successful cancerous clone survives and becomes clinically manifest. As the neoplasm is expanding and spreading, it adapts a different biology through selective pressure of its tissue environment. Although tumors are heterogeneous, they share the ability to proliferate beyond the growth of normal tissue. Loss of control of cell proliferation and survival therefore are key events. A better understanding of these molecular events will enable a more accurate diagnosis, therapy, and prognosis to be designed.

The literature on carcinogenesis already is complex beyond measure; however, a small number of understandable principles emerges. 41 According to Hanahan and Weinberg, 41 the clinical manifestation of a cancer is based on six essential alterations in cell physiology: self sufficiency in growth signals; insensitivity to growth inhibitory (antigrowth) signals; evasion of programmed cell death (apoptosis); limitless replicative potential; sustained angiogenesis; and tissue invasion and metastasis.

First, self sufficiency in growth signals: Normal cells require growth signals to make the transition from a quiescent to a proliferative cell. These signals, originating from growth factors, ECM components, and other molecules important for cell-cell interaction, are mediated via cell membrane receptors in normal cells. In contrast, tumor cells have the ability to proliferate in the absence of such extracellular stimuli. For example, many oncogenes can mimic normal growth signaling. The tumor cells are able to synthesize growth factors to which they are responsive themselves, such as PDGF or TGF-α23 which are known for various sarcomas. Overexpression of cell membrane-based growth factor receptors leads to the same effect. Overexpression of a receptor makes a cell hyperresponsive to low levels of such stimuli that usually would be below the threshold to elicit any cell proliferation. Furthermore, growth signaling autonomy originates from downstream cytoplasmic components, which integrate the signals emitted by growth factor receptors. The most important proponent is the Ras-Raf-MAPK cascade, leading to deregulation of growth signaling in the majority of tumors in humans. 45 Human epidermal growth factor receptor-2 (or erbB-2) recently was analyzed in patients with osteosarcoma. 70 It was shown that expression of HER-2/erbB-2 correlated with a worse event-free survival (47% versus 79% at 5 years) in patients with nonmetastatic disease and significantly less tumor necrosis after preoperative chemotherapy. 75 In addition, HER-2 (or erbB-2) also was correlated with clinically aggressive tumor growth and the promotion of metastatic potential in osteosarcoma. 75

C-Fos expression, an AP-1 transcription factor involved in mediating growth signals, was shown in transgenic mice to be necessary for the development of osteosarcoma. 103 Kakar et al 47 found a correlation of c-Fos expression with poor response to chemotherapy, where all eight patients with poor response were c-Fos positive, whereas only one patient was positive for c-Fos with a good response to chemotherapy. 47 Others found that c-Fos alterations occur more frequently in patients with recurrent osteosarcoma or metastatic disease. 81 Bone morphogenetic proteins, in particular the BMP Type II receptor, were found to correlate with metastasis in osteosarcoma, indicating a potential for growth stimulation in these tumors by the BMP family. 39

Second, insensitivity to growth-inhibitory (antigrowth) signals: Multiple antiproliferative signals act to keep a normal cell quiescent. Analogous to the positively acting counterparts, growth inhibitory signals are received by surface receptors that are coupled to intracellular signaling cascades. Basically, antigrowth signals can block proliferation either by forcing the cell into the G0 state of the cell cycle or by directly blocking the proliferation. Nearly all the antiproliferative signals converge to the retinoblastoma protein (pRb), which blocks the proliferation by altering transcription factors that govern the progression from G1 into the S phase. 104 Once the pRb-pathway is disrupted (either through mutation in the gene or through a signaling molecule), the cell becomes insensitive to antigrowth factors that block the advancement to the G1 phase under normal conditions. One of the best understood signaling molecules that inactivates pRb is TGF-β. Transforming growth factor-beta blocks the advance through G1 by suppression of c-myc or synthesis of p15INK4B and p21. 18 Disruption of the antigrowth mechanisms plays an important role in the majority of human cancers. Feugeas et al 24 analyzed 47 patients with primary osteosarcoma and found a loss of heterozygosity at the Rb locus in more than 60% of patients. The event-free survival rate at 5 years was 100% for patients without loss of heterozygosity compared with 43% for all patients with loss of heterozygosity, and 65% for patients with loss of heterozygosity who had nonmetastatic disease. Rb gene loss of heterozygosity therefore might be an early predictive feature for patients with osteosarcomas indicating a potentially unfavorable outcome. Similar results also were found by Wadamaya et al. 102 Rb gene alterations also were correlated with tumor grade, being more frequent in the higher grades. 106 Transforming growth factor-beta also has been associated with osteosarcoma. Franchi et al 32 found that high-grade osteosarcomas have a significantly higher expression of TGF-β1 than low-grade osteosarcomas and that TGF-β1 may be involved in determining the aggressive clinical behavior of high-grade osteosarcomas. Another isoform, TGF-β3 was shown to be related to disease progression in patients with osteosarcoma. 53 Preclinical studies also suggested a role of IGF-1 in the proliferation of osteosarcoma cells in vivo. 80 To the current authors’ knowledge, there is no report analyzing IGF-1 expression with correlation to clinical disease progression. However, it was found that the level of circulating serum IGF-1 is not predictive for the development of osteosarcoma. 85

Third, evasion of programmed cell death (apoptosis): An apoptotic program is present in latent form in all normal cells of the body. Tumor cells, however, acquire the ability to escape from programmed cell death, another hallmark of most human cancers. Basically, apoptosis can be divided in sensors and effectors. Sensors are responsible for monitoring the extracellular and intracellular environment for conditions of abnormality and normality, ultimately influencing the fate of each cell. Effectors include cell surface receptors that bind survival or death factors such as IGF-1, IGF-2, and TNF-α binding TNF-R1, respectively. 2,10 Independent of whether the signals eliciting apoptosis transduced via mitochondria or other intracellular pathways, the ultimate effectors are called caspases, which execute the death program leading to destruction of cellular structures, organelles, and the genome. 97 Therefore, changing components of the apoptotic pathway has an important role in the development of cancer. Resistance to apoptosis can be acquired by a mutation in the p53 tumor suppressor gene, removing a key component of the DNA damage sensor that induces the apoptotic effector cascade. 43 The PI3 kinase-AKT/PKB pathway is another way to transmit antiapoptotic survival signals, activated by IGF-1 and IGF-2. 22 Even though p53 has been reported 37,68,78 to be associated with poor prognosis with many human malignancies and poor response to chemotherapy, it has not been shown clearly for osteosarcoma, suggesting that p53 alone may not be a useful marker for disease progression. However, there is one report that implies that coexpression of p53 and p-glycoprotein is a strong indicator for a short survival. 78 One of the major problems in treating patients with osteosarcoma is drug resistance, which is explained by many factors. One of the best described models includes the plasma membrane protein p-glycoprotein, a product of the MDR1 gene, which is responsible for the efflux of many cytotoxic agents from the cell, including chemotherapeutic drugs. 107 Although there are controversial reports, a recent prospective multicenter study comprising 123 patients was unable to correlated the p-glycoprotein mRNA expression level with disease progression in patients with osteosarcoma. 107 It was thought that other genes may be more important in mediating drug resistance than p-glycoprotein. The role of the MDM2 oncogene is emerging. The MDM2 oncoprotein binds to the p53 suppressor protein and serves as a negative regulator of p53, thereby limiting the magnitude of p53 activation by DNA damaging agents. With respect to osteosarcoma, MDM2 gene amplification was associated with tumor progression and metastasis. 56 Alterations of the p53 and MDM2 pathway are frequent in osteosarcoma and usually are mutually exclusive events. 64 The protooncogene c-myc has several putative functions and is overexpressed in many neoplasms. In osteosarcoma, c-myc was found to be overexpressed in a high percentage of relapsed tumors and in metastases. If coexpressed with c-Fos, it is correlated strongly with the development of metastases. 34

Fourth, limitless replicative potential: All cells carry an intrinsic cell-autonomous program that limits their replicate multiplication, which operates independently of cell to cell signaling, as mentioned previously, and can be viewed as senescence. A successful tumor cell has to disrupt this pathway to acquire unlimited replicative potential. Telomeres, the ends of chromosomes, undergo a progressive shortening during each cell cycle in normal cells. 8 Once the progressive shortening reaches a critical threshold, the ends of the chromosomal DNA no longer are protected, which results in the death of the affected cell. 16 In tumor cells, however, telomere maintenance is virtually apparent in all types of cancer in humans. 9,90 In as many as 90%, the human reverse transcriptase telomerase is upregulated, adding hexanucleotide repeats to the ends of the telomeric DNA. Therefore, telomeres are maintained at a length above the critical threshold. This in turn permits unlimited multiplication of the cell. With respect to osteosarcoma in particular, there only are sparse and contradicting reports. Although Aue et al 4 concluded that the majority of osteosarcomas showed no telomerase activity, Scheel et al 89 concluded that telomere dysfunction might have major implications in tumor progression in patients with osteosarcoma.

Fifth, sustained angiogenesis: Normal cells reside within 100 μm of a capillary blood vessel to receive an optimal supply of oxygen and nutrients. Because proliferating cells within a tissue do not have the capability to initiate blood vessel growth, tumor cells have to develop angiogenic ability. 40 Angiogenesis can be stimulated or blocked by positive or negative signals. The first group comprises VEGF and FGF1 and FGF2, which bind to tyrosine kinase receptors on endothelial cells. 101 Thrombospondin-1, which binds to CDC36, a transmembrane receptor that is coupled to intracellular Srclike tyrosine kinases, is an inhibitor of angiogenesis. Currently, the number of inducers and inhibitors of angiogenesis still is increasing. The ability to induce or sustain angiogenesis from vascular quiescence is known as the angiogenic switch. Tumors have the ability to activate this switch by changing the balance of inducers and inhibitors. 40 The mRNA expression of VEGF in osteosarcoma has been reported. 59 All 30 specimens that were analyzed expressed VEGF, and patients with the isoforms VEGF165 had a poorer prognosis compared with patients who did not express VEGF165. The authors concluded that this isoform might be essential for neovascularization in osteosarcoma. Kaya et al 50 found VEGF mRNA expression in 63% of untreated osteosarcomas. Strikingly, there was a strong correlation between expression of VEGF and the development of pulmonary metastases (89% of VEGF-positive tumors versus 10% of VEGF-negative tumors), but also for disease-free and overall survival.

Sixth, tissue invasion and metastasis: Cancer cells have the ability to invade and metastasize to new sites in the body. Metastases are the cause of death in 90% of humans with cancer. 91 In metastasizing tumor cells, many proteins show altered function according to the changing tissue microenvironment. These include cell-cell adhesion molecules such as cadherins and integrins, which link the cells to the ECM. For example, tumor cells facilitate invasion by shifting their expression of integrins from those that favor the ECM to other integrins, which bind stromal components produced by proteases. 99 Furthermore, metastasis also involves extracellular proteases. 12 It is thought that proteases on the cell surface facilitate invasion by cancer cells through normal epithelial layers and across vessels. However, the complexity originating from multiple tissues expressing different proteases is not yet well understood. The activation of proteases and the altered binding specificities of cadherins, cell-cell adhesion molecules and integrins play a key role in metastasis and invasion. Recently, a molecular answer was provided as to why tumor cells might have a preference for certain organs. Soluble factors called chemokinases are released in large quantities from certain organs, which have the capacity to attract circulating breast cancer cells to take up residence there. 72 With respect to osteosarcoma, anomalous cadherin expression was related to metastasis 48 and c-MET 73 and tenascin-C. 94 As mentioned previously, c-Fos alterations have been shown to occur more frequently in patients with osteosarcoma who have recurrent or metastatic disease. 82 Obviously, the understanding of the etiology of osteosarcoma is not complete. It is apparent that numerous initiating factors are known and presumably others are yet to be found. These factors may cause genetic alterations, which ultimately result in abnormal, unregulated cell cycling. Many new insights have emerged from various sources; the wealth of new information generated by molecular analysis makes clear that a new door is opened with the potential of significantly improving patient care in the future.

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Glossary

  • AP-1 = Activator Protein-1, a member of transcription factor that is activated on stress and facilitates transcription of target genes.
  • BME = Bloom’s syndrome gene, located on chromosome 15q26.1 encoding for a DNA helicase.
  • c-Met = The hepatocyte growth factor receptor is a tyrosine kinase receptor and has been causally implicated with tumor progression and metastasis, particularly in colon carcinoma. Activation of these receptor-tyrosine kinases can stimulate numerous of specific pathways directly effecting tumor cell migration, survival, and proliferation. The aberrant regulation of the receptor-tyrosine kinases often is seen in advanced disease and affects significantly the metastatic phenotype of tumor cells.
  • Fos = Fos together with Jun family proteins function as dimeric transcription factors that bind to AP-1 regulatory elements in the promoter and enhancer regions of numerous mammalian genes. They participate in the regulation of various cellular processes including cell proliferation, differentiation, apoptosis, and oncogenesis.
  • G1/S-checkpoint = One of the four control mechanisms during the cell cycle that ensures that chromosomes are intact and the cell cycle is completed before the following stage is initiated.
  • MDM2 = Protooncogene discovered by its genomic amplification on a murine double minute chromosome, it inhibits p53 through either ubiquitin-dependent p53 degradation in the cytoplasm or repression of p53’s transcriptional activity in the nucleus.
  • MDR1 gene = Encodes for the plasma membrane protein p-glycoprotein. This is thought to facilitate the efflux of cytotoxic agents from the cell.
  • p15INK4B = Cyclin-dependent kinase inhibitor gene, which is transcribed actively after TGF-β exposure. Methylation of the p15INK4B gene is significantly correlated with blastic bone marrow involvement, and sequential analyses have shown that methylation increases with disease evolution toward AML.
  • p21 = Protein that plays a critical role in regulating cell growth and cell response to DNA damage. Its primary targets are the cdk-cyclins, which regulate the progression of eukaryotic cells through the cell cycle, and proliferating cell nuclear antigen (PCNA). p21 forms complexes with a class of cdk-cyclins to inhibit their kinase activity and with PCNA to inhibit DNA synthesis. Cell cycle arrest in G-1 (G-1 checkpoint) after DNA damage is mediated by p53. p53 upregulates p21 expression in response to DNA damage, which in turn inhibits cdk2-associated kinase activity.
  • PI3-kinase-AKT/PKB = Phosphoinositide 3-kinases phosphorylate the 3′-OH position of the inositol phospholipids, producing three lipids. These lipids control the activity and subcellular localization of a diverse array of signaling transduction molecules. These molecules are activated by various extracellular stimuli and have been implicated in a wide range of cellular processes, including cell adhesion and cell survival.
  • Ras-Raf-MAPK = Network of interacting proteins that governs a large number of cellular processes, mediating the transmission of extracellular signals to their intracellular targets by sequential stimulation of several cytoplasmic protein kinases to initiate cellular processes such as proliferation, differentiation, and development.
  • Rb gene = Encodes the Rb protein, which arrests cells in the G1 phase of the cell cycle. Rb is phosphorylated (inactive form, predominating in proliferating cells) and dephosphorylated (active form, abundant in quiescent or differentiating cells). It plays a fundamental role in cellular regulation and is a target of tumorigenic mutations in many cell types.
  • RECQL4 = Gene located on chromosome 8q24.3 encoding a DNA helicase that unwinds double-stranded DNA into single-stranded DNA. Mutation in this gene causes Rothmung-Thomson syndrome.
  • Tenascin-c = Is a modular and multifunctional extracellular matrix glycoprotein that is regulated exquisitely during embryonic development and in adult tissue remodeling. Tenascin-C gene transcription is controlled by intracellular signals that are generated by multiple soluble factors, integrins, and mechanical forces.
  • TP53 = Gene encodes for the protein p53 of approximately 53 kDa in size. It mediates a major tumor suppression pathway that frequently is altered in human cancers. p53 is inhibited during normal cell growth by MDM2. p53 is activated after DNA damage through p53 phosphorylation or in response to oncogenic insults by the activation of ARF, a tumor suppressor encoded by the alternative reading frame of the INK4a locus.
  • (v- or c-) src = The nonreceptor tyrosine kinase Src has important roles in many aspects of cell physiology. The viral src gene was the first retroviral oncogene to be identified, and its cellular counterpart was the first protooncogene to be discovered. Src in general was an important entry point into the molecular genetics of cancer.
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Section Description

Mark T. Scarborough, MD; B. Hudson Berry, MD; W.F. Enneking, MD; Albert Aboulafia, MD; and Eugene Mindell, MD, Guest Editors

© 2002 Lippincott Williams & Wilkins, Inc.