- Email: [email protected]
Thrombospondins and tumor angiogenesis
Review
TRENDS in Molecular Medicine Vol.7 No.9 September 2001
401
Thrombospondins and tumor angiogenesis Florence de Fraipont, Ainsley C. Nicholson, Jean-Jacques Feige and Erwin G. Van Meir The thrombospondins (TSPs) are a family of five secreted proteins that are widely distributed in the extracellular matrix of numerous tissues. TSPs are multimodular and each domain specifies a distinct biological function through interaction with a specific receptor. TSP1 and TSP2 have anti-angiogenic activity, which, at least for TSP1, involves interaction with the microvascular endothelial cell receptor CD36. Expression of TSP1 and TSP2 is modulated by hypoxia and by oncogenes. In several tumors (thyroid, colon, bladder carcinomas), TSP1 expression is inversely correlated with tumor grade and survival rate, whereas in others (e.g. breast carcinomas), it is correlated with the stromal response and is of little prognostic value. Recent studies suggest that TSPs or TSP-derived peptides retaining biological activity could be developed into promising new therapeutic strategies for the anti-angiogenic treatment of solid tumors.
Florence de Fraipont Jean-Jacques Feige INSERM EMI 0105, Dept of Molecular and Structural Biology, Commissariat à l’Energie Atomique, Grenoble, France. Ainsley C. Nicholson Erwin G. Van Meir* Laboratory of Molecular Neuro-Oncology, Neurosurgery Dept and Winship Cancer Institute, Emory University, Atlanta, GA, 30322, USA. *e-mail: [email protected]
Platelet thrombospondin (now named TSP1) is the canonical member of a family of structurally related proteins with five distinct members, and was named to reflect its initial purification from thrombin-activated platelet releasates. TSP1 was later shown to be secreted from a wide variety of epithelial and mesenchymal cells. All five TSP family members are multimeric and multimodular heparinand calcium-binding proteins, but two subfamilies can be distinguished1–3. TSP1 and TSP2 have a similar structural organization, as shown in Fig. 1. Each subunit of these trimeric molecules is composed of an N-terminal heparin-binding domain, a linker domain enclosing the two cysteine residues implicated in trimerization, a procollagen-homology domain, three properdin-like type I repeats, three epidermal growth factor (EGF)-like type II repeats, seven calcium-binding type III repeats and a globular C-terminal domain. The remaining TSP proteins, TSP3, TSP4 and TSP5/COMP, resemble one another but differ from TSP1 and TSP2 in that they are pentameric, and their subunits possess four instead of three type II repeats and lack the procollagenhomology domain and the three type I repeats (Fig. 1). Regulation and expression patterns of the TSP family members differ. During murine development, TSP1 is expressed in liver, kidney, and gut, and TSP2 is expressed primarily in connective tissues4. In adult tissues, TSP1 and TSP2 are expressed in a variety of organs and have overlapping but distinct expression patterns5–7. Other members are particularly abundant in cartilage and bone tissues. A variety of cellular receptors that recognize specific peptide domains of TSP1 have been identified (Fig. 2). They appear to mediate a variety of biological functions8. CD47/IAP (integrin-associated protein) binds to the C-terminal domain, integrins αvβ3 and http://tmm.trends.com
αIIbβ3 bind to RGD motifs in the last type III repeats, and binding of the scavenger receptor LRP (LDL receptor-related protein) is dependent upon association of TSP1 with heparan sulfate proteoglycans (HSPGs). Recently, a sequence in the N-terminal domain was also found to bind integrin α3β1 on tumor cells and aortic endothelial cells9. Most interestingly, discrete repetitive sequences (comprising CSVTCG and adjacent sequences) present in the second and third type I repeats mediate the binding of TSP1 to CD36 on endothelial cells, resulting in inhibition of angiogenesis3,10. Although all these sequences are well conserved in TSP2, only the HSPG binding and the HSPG-mediated interaction with LRP has been clearly demonstrated using the full-length TSP2 protein11. Almost 70 proteins in the human genome also possess type I repeat motifs (Ainsley C. Nicholson and Erwin G. Van Meir, unpublished). A subset of these affect angiogenesis (Fig. 3). Brain angiogenesis inhibitor (BAI)-1 and ADAMTS-1 and -8 – two members of the ADAMTS (a disintegrin and metalloproteinase with TSP motifs) family – inhibit angiogenesis, whereas connective tissue growth factor (CTGF), which possesses a single type I repeat, stimulates angiogenesis12. By contrast, several proteins containing type I repeats have no angiogenic effects. These include complement component proteins from the alternate immune response pathway (including C6, C7, C8, C9), properdin, the neurotrophic factors F-spondin, SCO-spondin, semaphorins 5A and 5B and several other ADAMTS proteins12. Most proteins containing TSP-type I repeats have an affinity for heparin, but the elements within type I repeats that might confer pro- or anti-angiogenic activity are still being determined. It should be noted that, despite the absence of TSPs from the genomes of non-vascularized invertebrate metazoans such as Caenorhabditis elegans or Drosophila melanogaster, some proteins of these species do contain type I repeat motifs, indicating that this functional motif arose early and has been preserved through evolution13. TSP1 and TSP2 are anti-angiogenic
Our current appreciation of the involvement of TSPs in diverse biological processes extends far beyond the initial observations of their roles in platelet aggregation and coagulation2,14. TSP1 released by activated platelets participates in the formation and resolution of the fibrin clot, by binding to fibrin, plasminogen and urokinase through its N-terminal
1471-4914/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1471-4914(01)02102-5
Review
402
Fig. 1. Members of the thrombospondin (TSP) family have a multimodular structure. TSP1 and TSP2 are composed of an N-terminal heparin-binding domain (large green circle), a procollagen homology domain (dark-blue oval), three type I repeats (green circles), three epidermal growth factor (EGF)-like type II repeats (orange diamonds), seven calcium-binding type III repeats (pale blue rectangles) and a C-terminal globular domain (blue circle). By contrast, TSP3, TSP4 and TSP5/COMP contain distinct N-terminal domains (light-blue, red, purple circles), four EGFlike repeats, seven type III repeats and a similar C-terminal globular domain. ‘S’ indicates the position of the cysteine residues involved in multimerization. Sequence homology increases from the N- to the C-termini of these proteins.
TRENDS in Molecular Medicine Vol.7 No.9 September 2001
TSP1 S
COOH
NH2 S TSP2 S S TSP3 S S TSP4 S S TSP5/COMP S S
TRENDS in Molecular Medicine
heparin-binding domain. TSP1 also participates in the formation of molecular bridges between platelets and inflammatory leukocytes through interactions with cell-surface receptors such as CD36 or the αvβ3 and αIIbβ3 integrins. The regulatory role of TSPs on endothelial cell adhesion, migration and proliferation has been intensely studied in vitro since the late 1980s15–18. Human platelet TSP1 specifically inhibits both the proliferation of endothelial cells from various tissue origins and their three-dimensional organization into capillary-like structures when grown in collagen gels. TSP1 was also found to inhibit neovascularization in vivo and endothelial cell migration toward fibroblast growth factor (FGF)-2 in vitro (ED50 ~1 nM)17,19. However, it should be noted that, at
Trimerization
Latent TGFβ activation RFK
S COOH
NH2 S WXXW CSVTCG
QNV α3β1
HSPG
Cell adhesion Hemostasis
CD36 Anti-angiogenesis cell adhesion
RGD
RFYVVM IRVVM
αvβ3
IAP/CD47 αIIbβ3 Cell adhesion Cell adhesion
LRP gp330 Internalization / degradation TRENDS in Molecular Medicine
Fig. 2. Multiple receptors recognize thrombospondin (TSP)1. Almost every domain of the TSP1 molecule binds to a specific receptor. The peptide sequences indicated below each module are specifically recognized by the receptors indicated in oval boxes. Two distinct domains of the TSP1 molecule bind heparan sulfate proteoglycans (HSPG), and this binding appears to be a prerequisite for binding to the scavenger receptor LRP. The biological function of each receptor is indicated below each box. Abbreviations: IAP, integrin-associated protein; LRP, low-density lipoprotein receptor-related protein; TGFβ, transforming growth factor β.
http://tmm.trends.com
higher concentrations (20–100 nM), TSP1 promotes endothelial cell migration19,20. Using proteolytic fragments and synthetic peptides, the angio-inhibitory activity of TSP1 has been mapped to the procollagen homology domain and to the type I repeats19. Two separate non-overlapping peptides [one of them containing a latent transforming growth factor (TGF)β-activating sequence21] derived from the second and third type I repeats displayed anti-angiogenic activity. However, the anti-angiogenic activity of TSP1 appears to function independently from its activation of latent TGFβ (Ref. 21). TSP2 also contains type I repeats, and recombinant mouse TSP2 and natural bovine TSP2 have been shown by in vitro assays to inhibit angiogenesis to a similar degree as TSP1, whereas TSP5/COMP, a pentameric member of the TSP family lacking type I repeats, is inactive18. The anti-angiogenic action of the CSVTCGcontaining type I repeats in TSP1 is mediated by the interaction with the membrane receptor CD36 present on the surface of blood capillaries. As shown in Figure 4, this leads to the sequential activation of cytoplasmic tyrosine kinase p59fyn, caspase-3-like proteases and p38 mitogen-activated protein (MAP) kinase and results in endothelial cell apoptosis10. Endothelial cells exposed to TSP1 display increased levels of Bax expression, decreased levels of Bcl-2 expression, and process caspase-3 into smaller pro-apoptotic forms22. However, other investigators found a pro-angiogenic activity for TSP1. Nicosia and Tuszynski75 observed that the presence of TSP1 in serum-free cultures of aortic ring explants in a collagen gel promoted the number and length of microvessels outgrowing the explants. As observed with cell migration, this effect was observed at high TSP1 concentrations (ED50 ~20–40 nM) and might result from the interaction of TSP1 domains other than those shown to have anti-angiogenic activity with a distinct receptor. In accordance with this hypothesis, Taraboletti et al.23 recently reported that the N terminal heparin-binding domain of TSP1 is angiogenic in the rabbit cornea assay, whereas the 140-kDa fragment lacking this domain appears to inhibit the FGF2-driven angiogenic response. The activity of the intact TSP proteins and their derived peptides might be very dependent on their conformation. It was recently shown that engagement of the α3β1 integrin by a soluble TSP-1 peptide inhibits endothelial cell proliferation and angiogenesis, whereas the same peptide stimulates endothelial cell proliferation when immobilized9. The role of TSP1 and 2 in embryonic angiogenesis has been addressed in several animal studies. During mouse embryonic development, TSP1 mRNA is detected in capillaries of day 16–18 embryos4. However, disruption of the TSP1 gene in mice does not appear to dramatically alter the normal embryonic development of the vasculature, as these animals are viable and appear to develop normally24. TSP2 is expressed in capillaries and large vessels
Review
Fig. 3. Several proteins regulating angiogenesis possess thrombospondin (TSP)-like type I repeats. TSP type I repeats (small green circles), which govern the angiostatic activity of thrombospondin-1, are found in several proteins that affect angiogenesis. A disintegrin and metalloproteinase with TSP repeats (ADAMTS)-1 and -8 and brain angiogenesis inhibitor (BAI)-1 are angiostatic, whereas connective tissue growth factor (CTGF) promotes angiogenesis. Other known proteins containing type I repeats have not been shown to affect angiogenesis.
TRENDS in Molecular Medicine Vol.7 No.9 September 2001
S
TSP1
403
TSP1
S
Endothelial cell ADAMTS-1,8
CD36
P
p38
p59fyn
Nucleus
Apoptosis
Caspases (3-like)
P BAI1 Caspases (3-like)
pp38 Angiogenesis
CTGF TRENDS in Molecular Medicine TRENDS in Molecular Medicine
from day 10 post-conception continuously through parturition on day 18 (Ref. 4). The phenotype of TSP2-knockout mice is more dramatic than that of TSP1 knockouts25. Although viable, these mice develop alterations in the tensile strength of skin and tendons, an abnormal bleeding time and an increased density of blood vessels in many tissues. This last feature is a clear indicator of the importance of the role of TSP2 in the regulation of organ vascularity. Expression and regulation of TSP1 and TSP2 Regulation by hypoxia
Hypoxia strongly favors angiogenesis through induction of vascular endothelial growth factor (VEGF) expression and improved VEGF mRNA stability26. It was therefore of interest to determine whether hypoxia also affects TSP1 expression. In endothelial cells (HUVEC or HMVEC), hypoxia was reported to induce TSP1 gene and protein expression by post-transcriptional stabilization of the TSP1 mRNA27. By contrast, hypoxia strongly decreases TSP1 mRNA levels in both p53+/+ and p53−/− human and rodent fibroblasts and in several human glioblastoma tumor cell lines28,29. No significant effect was observed in human cervical epithelial cells28, and TSP1 expression was unaffected by hypoxia in bovine adrenocortical cells (but TSP2 mRNA and protein levels were downregulated) (M. Keramidas and J.J. Feige, personal communication). These observations suggest that the regulation of TSP1 and TSP2 expression by hypoxia could depend on tissue type, cell transformation, and experimental conditions. Although downregulation of TSP1 by tumor cells appears to promote angiogenesis, upregulation by endothelial cells could be part of a negative feedback loop, although this remains to be established. Furthermore, it is unclear whether downregulation by hypoxia is a specific regulatory mechanism or the result of decreased metabolic activity under hypoxia. Regulation by oncogenes and tumor-suppressor genes
The initial observation that TSP1 possesses anti-angiogenic activity, and the later discovery that genetic alteration of certain tumor suppressor genes http://tmm.trends.com
Fig. 4. Thrombospondin (TSP)-1 induces endothelial cell apoptosis. TSP1 binds through its type I repeats to CD36 receptors present at the surface of microvascular endothelial cells and activates the cytoplasmic protein tyrosine-kinase p59fyn. This kinase in turn activates caspase-3-like proteases, which activate p38 mitogen-activated protein kinase. Translocation of the phosphorylated form of p38 mitogen-activated protein kinase into the nucleus induces expression of caspase-3 resulting in endothelial cell apoptosis. Adapted from Ref. 10.
correlates with simultaneous loss of TSP1 expression and acquisition of an angiogenic phenotype by tumor cells16,17 suggests an exciting new paradigm for tumor progression. Among tumor suppressor loci affecting TSP1 expression, TP53 is the best characterized. The TP53 tumor suppressor gene is mutated in over 50% of human cancers. Spontaneous loss, after several passages in culture, of the wild-type (wt) TP53 allele from human Li-Fraumeni fibroblasts with a TP53 wt/mutant genotype was accompanied by a simultaneous decrease in the secretion of TSP1, whereas TSP2 expression remained stable. Reintroduction of the wt TP53 gene into these fibroblasts restored TSP1 mRNA levels30,31, confirming that the reduction in TSP1 expression was causally related to the loss of p53 activity. Such regulatory mechanisms might be tissue-specific, as modulation of exogenous p53 did not modify cellular TSP1 levels in the p53-null human glioma cell line LN-Z308 (Refs 29,32). Analysis of patients with invasive transitional cell carcinoma of the bladder determined that reduced TSP1 expression correlates with the presence of p53 alterations in tumors and with increased recurrence rates, increased microvessel density, and decreased overall survival33. Such a clear-cut association might not be a general feature for all tumor types, as analysis of certain cancers, including lung cancer, cholangiocarcinoma, and glioblastoma, found no association between the presence of p53 alterations and TSP1 mRNA expression29,34,35. Oncogenes were also found to regulate TSP1 expression in the Li-Fraumeni model described above. Introduction of activated ras resulted in cell transformation and a further decrease in TSP1 mRNA levels31. Inhibition of basal TSP1 expression by various oncogenes or oncogenic signals including not only ras but also v-src, v-myc, polyoma middle T antigen and overexpressed c-jun has been reported36–41. However,
404
Review
TRENDS in Molecular Medicine Vol.7 No.9 September 2001
this is not a universal feature of oncogenes, as SV40 large T or BPV1 did not inhibit TSP1 expression37. Thus, research has demonstrated that both oncogene activation and loss of tumor-suppressor gene expression are directly or indirectly associated with a decrease in TSP1 expression. This could increase the likelihood of the tumor gaining the capacity to induce blood vessel formation, thereby making the ‘angiogenic switch’ and progressing towards greater malignancy. Expression of TSP1 and TSP2 in human cancers
Several studies have demonstrated that the extent of tumor angiogenesis is related to clinical outcome42. As TSP1 and TSP2 inhibit angiogenesis, and TSP1 expression can be downregulated by oncogene activation or tumor suppressor loss, it has been hypothesized that their expression could be inversely correlated with tumor progression, recurrence or metastatic potential (see Table 1). The results of several studies aimed at determining TSP1 levels in different tumor types, by Northern-blot, RT-PCR or immunohistochemistry, are reported in Table 1. In evaluating these results, it is important to differentiate between expression of TSP1 and/or TSP2 by tumor cells, and expression in stromal tissues that surround a tumor. This can only be achieved by immunohistochemical or in situ hybridization analyses. In several distinct tumors including breast, cholangial, esophageal, gallbladder, thyroid and bladder carcinomas, TSP1 was present in the stroma and weakly expressed or absent in the tumor cells33,34,43–46. Many other studies using RT-PCR or biochemical methods to detect TSPs did not address the question of stromal versus tumoral distribution but rather determined an overall content in the analyzed tissue specimens. However, from the results of such studies, one can differentiate several tumors in which TSP1 or TSP2 expression is inversely correlated to tumor grade and others in which TSP1 or TSP2 expression is higher in carcinomas than in benign tumors or normal tissue. Human tumors in which increased TSP1 or TSP2 expression is associated with malignancy
In breast tumors and cholangiocarcinomas, the stromal expression of TSP1 was much stronger in tumors than in normal tissue34,43,47,48. Similarly, in colorectal carcinomas and pleural mesothelioma, the level of TSP1 expression was higher in tumors than in normal tissue49,50. In all of these tumors, TSP1 levels were of little prognostic value. By contrast, in esophageal and gallbladder carcinomas, TSP1 expression appeared to be directly correlated with the occurrence of metastases44,45. Human tumors in which decreased TSP expression is associated with malignancy
In human adrenocortical carcinomas, non-small cell lung carcinomas, gliomas and thyroid carcinomas, http://tmm.trends.com
TSP levels were lower in malignant tumors than in adenomas29,46,51–54. Several studies showed that TSP1 expression is inversely correlated with tumor grade and survival rate in thyroid, colon and bladder carcinomas33,46,55. In non-small cell lung carcinoma, only TSP2 expression (not TSP1) appeared as a significant prognostic marker52. Similarly, increased TSP1 expression by tumor cells was found to correlate with reduced metastatic potential in experimental tumors formed from melanoma, lung, and breast carcinoma cell lines41. One possible explanation for the different effects of TSP1 expression in various tumor types relates to its interaction with TGFβ. TSP1 binds to the latent precursor of TGFβ via the RFK sequence (located between the first and the second type I repeats) and the WXXW sequences. This favors the release of active TGFβ, either directly21 or indirectly56,57. The importance of this interaction is suggested by the similarity of histological abnormalities in nine organ systems between TGFβ null and TSP1 null mice58, although other physiological latent TGFβ activation mechanisms exist59. TGFβ triggers apoptosis in several tumor-cell types, and might also contribute to tumor growth by stabilizing the tumor vasculature60. In cancer formation, the release of active TGFβ can result in upregulation of uPA (urokinase plasminogen activator), uPA-receptor and PAI-1 (plasminogen activator inhibitor-1), three components of the proteolytic system that promotes malignant invasiveness61. This promotion of malignancy by interaction with TGFβ acts in opposition to the anti-tumorigenic/anti-angiogenic activities of TSP1. The overall effect of TSP1 overexpression on tumor cells could, therefore, reflect the balance between anti-angiogenic forces versus invasive forces. For a specific tumor, whether TSP overexpression promotes or inhibits malignancy could be at least partially dependent on the concentration of TGFβ at the tumor site. Plasma levels of TSP1
Plasma TSP1 levels in patients with metastatic breast, lung or gastrointestinal carcinomas were found to be two- to three-fold higher than those of healthy volunteers or patients with non-metastatic disease62. A direct correlation was established between plasma TSP1 level and the stage of disease in patients with gynecologic or colorectal malignancies63,64. As the origin of plasma TSP1 (platelet clotting, endothelial cells or tumor cells) has not been completely characterized, and the function of circulating TSP1 in cancer patients is unknown, it is difficult to determine whether high plasma TSP1 concentrations are beneficial in reducing growth of distant metastases for patients with certain tumor types, or produce a detrimental increase in metastatic invasiveness. It is also not clear whether plasma TSP1 levels can be used as a prognostic factor.
Review
TRENDS in Molecular Medicine Vol.7 No.9 September 2001
405
Table 1. Correlations between thrombospondin (TSP) levels and clinical features in different human cancers Tumor type
Cohort Method size
Results
Refs
Bladder carcinoma
163
Immunohistochemistry
TSP1 expression is inversely correlated with disease recurrence and overall survival
33
Breast carcinoma
101
Radioimmunoassay
High levels of TSP1 found in carcinomas; absence of correlation with clinical parameters
48
25
Immunohistochemistry and Weak TSP1 staining in normal tissue; strong staining in stroma around in situ hybridization tumor cells
43
3
Northern blot
TSP1 is inversely correlated with malignant progression
41
33
RT-PCR
TSP1 and TSP2 expressions are higher in invasive carcinomas than in normal or benign tissue; no correlation with clinical parameters or vascularity
47
Colorectal carcinoma 61
RT-PCR
TSP2 expression is inversely correlated with the occurrence of distant metastases; no correlation of TSP1 expression with clinical parameters
55
62
RT-PCR
TSP2 is more frequently expressed in tumoral than in extraneoplastic colon mucosa; TSP1 expression not significantly different
50
100
Immunohistochemistry
TSP1 expression is inversely correlated with good prognosis
74
Cholangiocarcinoma 11
Northern blot and immunohistochemistry
TSP1 expression is higher in tumoral than in surrounding healthy tissue; no correlation with clinical parameters
34
Adrenocortical carcinoma
43
ELISA
Lower TSP1 concentrations in carcinomas and transitional tumors than in adenomas
51
Esophageal squamous cell carcinoma
54
Immunohistochemistry
Higher TSP1 expression in tumoral than in healthy tissue; TSP1 immunoreactivity detected in the stroma, infrequently in the cancer cells; higher incidence of lymph node metastasis in the TSP1-positive cases
44
Gallbladder carcinoma
53
RT-PCR and immunohistochemistry
TSP1 immunoreactivity detected in the stroma, infrequently in the cancer cells; TSP1 expression is correlated with lymph node metastasis
45
Glioma
17
Immunohistochemistry
Strong TSP1 staining in normal brain, in low-grade astrocytomas and in anaplastic astrocytomas; weak or no staining in high-grade glioblastomas
53
37
RT-PCR
TSP2 gene expression is inversely associated with histological grade; no correlation of TSP1 expression with clinical parameters
54
10
Immunohistochemistry
Of recurring gliomas in which TSP1 expression was found, 4/5 had decreased TSP1 expression in higher-grade gliomas
29
Non-small cell lung carcinoma
78
RT-PCR
Decreased TSP2 expression and inverse correlation with good prognosis; no correlation of TSP1 expression with clinical parameters
52
Malignant pleural mesothelioma
78
RT-PCR
TSP1 is overexpressed but its expression has little prognostic value
49
Thyroid carcinoma
84
RT-PCR and immunohistochemistry
Downregulation of TSP1 expression in aggressive carcinomas
46
Anti-angiogenic thrombospondins in cancer therapy
Since the discovery of the role of angiogenesis in the pathogenesis of tumor growth and metastasis, new cancer treatment strategies exploiting selective inhibition of tumor neovascularization have been explored65. In this context, TSP1 and TSP2 have been tested as anti-tumor agents in several xenograft models. Animal models in which TSP1 or TSP2 decreases tumor growth
Transfection of glioblastoma, fibrosarcoma, breast carcinoma and cutaneous carcinoma cells with a plasmid expressing the TSP1 protein results in reduced tumor growth in immunocompromised nude (nu/nu) mice29,66–69. Similar results were observed when squamous epithelial carcinoma cells were transfected with a TSP2 expression plasmid70. Moreover, in this model, TSP2 was significantly more active than TSP1 and had synergistic antitumoral http://tmm.trends.com
effects with TSP1 that resulted in complete suppression of tumor development70. In all these models, inhibition of tumor growth was generally accompanied by a decrease in tumor vascularization confirming the hypothesis that angiostatic factors could be used to inhibit tumor growth. Impressive inhibition of tumor growth with potentially angiostatic peptides from TSP1 was observed in intracerebral rat glioma models (C6, 9L)71. Surprisingly, a difference in vascular density could not be detected when in vivo imaging was used71, perhaps because of the limited resolution of this technique (250–500 µm range), suggesting that the methods chosen for precise visualization of the capillary networks are critical. Systemic release of high levels of TSP1 can also inhibit angiogenesis. Expression of high levels of TSP1 by a human fibrosarcoma line implanted subcutaneously in nude mice reduced growth of experimental melanoma metastases in the lung of the
406
Review
TRENDS in Molecular Medicine Vol.7 No.9 September 2001
same animals. Apparently, systemic TSP1 released from the fibrosarcoma prevented the metastases from establishing vascularization, thereby reducing their growth. This effect could be recapitulated by direct injection of purified TSP1 protein67. Animal models in which TSP1 augments tumor growth
Other animal models, however, cast doubt on the anti-tumorigenic potential of TSP1. Although overexpression of TSP1 by breast cancer cells resulted in smaller tumors than controls, overexpression in the same cell of a TSP1 protein fragment lacking the last 98 C-terminal residues resulted in tumors that grew larger than control tumors. This was surprising, because the truncated TSP1 had retained previously identified angiostatic domains66. Similarly, cancer progression was inhibited by neutralization of TSP1 expression in squamous carcinoma by anti-sense mRNA (Ref. 72) and in breast tumors by a polyclonal anti-TSP1 receptor antibody73. These observed differences in effect of TSP1 expression on tumor growth might be more fully explained when we have a better understanding of the specific functions of TSP1 in various cell types. Clinical trials with TSP1 and TSP2 Acknowledgements E.G.V.M. was supported by grants from the NIH (NS 41403 and CA86335), the Swiss National Science Foundation (4037-044729), and the University Research Committee of Emory University. A.C.N. was supported by NIH training grant T32 NS07480. F.d.F. and J.J.F. were supported by INSERM (EMI 0105), CEA and grants from the Ligue Nationale contre le Cancer (Comités de l’Isère et de la Drôme) and the GEFLUC.
Because of their large size (> 450 kDa), TSP1 and TSP2 have not been used in clinical trials. Peptides derived from TSP1 type I repeats, the region which is critical for inducing endothelial cell apoptosis after engaging the CD36 receptor10, might have clinical applications. Methods for stabilizing these peptides in vivo and delivering them to tumor sites are currently being researched, and will be essential in the development of cancer treatments based on TSP1 or TSP2. Conclusion
Of the five members of the TSP family of proteins, only TSP1 and TSP2, which contain type I repeat motifs, display anti-angiogenic activity. The
References 1 Bornstein, P. and Sage, E.H. (1994) Thrombospondins. Methods Enzymol. 245, 62–85 2 Feige, J-J. (2000) Thrombospondins. In Encyclopedic Reference of Vascular Biology and Pathology (Bikfalvi, A., ed.), pp. 285–292, Springer 3 Lawler, J. (2000) The functions of thrombospondin-1 and -2. Curr. Opin. Cell. Biol. 12, 634–640 4 Iruela-Arispe, M.L. et al. (1993) Differential expression of thrombospondin 1, 2, and 3 during murine development. Dev. Dyn. 197, 40–56 5 Corless, C.L. et al. (1992) Colocalization of thrombospondin and syndecan during murine development. Dev. Dyn. 193, 346–358 6 Kyriakides, T.R. et al. (1998) The distribution of the matricellular protein thrombospondin 2 in tissues of embryonic and adult mice. J. Histochem. Cytochem. 46, 1007–1015 7 Danik, M. et al. (1999) Bovine thrombospondin-2: complete complementary deoxyribonucleic acid http://tmm.trends.com
8 9
10
11
12
regulation of TSP1 expression by tumor suppressors and oncogenes not only implies that the angiogenic switch during tumor formation is facilitated by loss of TSP1 expression, it also suggests possible uses for the anti-angiogenic properties of these proteins, or TSP-derived peptides retaining anti-angiogenic activity, in cancer therapy. Although the differing results of various studies correlating levels of TSP expression with tumor progression and clinical prognosis is perplexing at this time, they do indicate a complexity of interaction between TSPs and various cell types. These differences could be caused by as yet undiscovered receptors, or by varying responses within the different cell types to stimulation by TSPs. This could limit the usefulness of anti-tumor gene therapy using TSPs to certain tumor types. It should be noted, however, that in tumor types that do respond to TSP stimulation, the anti-tumorigenic response is dramatic. This certainly encourages continued research into the use of TSPs, particularly TSP1 and TSP2, as anti-cancer agents. Future investigations are expected to reveal the molecular basis for the differing effects of TSP1 on tumorigenesis in different tumor types, and to describe the molecular pathways for the regulation of TSP1 by multiple tumor suppressors and oncogenes. Research into the possibility of using TSP1 or TSP2 as prognostic markers for cancer patients could improve the precision of clinical predictions. Cancer therapies using TSPs might be developed, and it will be particularly interesting to learn if TSP type I repeat peptides or peptide mimetics can be utilized in clinical therapy and how they compare with other anti-angiogenic factors such as TNP-470, angiostatin, and endostatin. There are issues to resolve before the anti-angiogenic properties of TSP1 and TSP2 can be employed for the treatment of cancer, but substantial progress has already been made, and the intense research in this field promises many new discoveries.
sequence and immunolocalization in the external zones of the adrenal cortex. Endocrinology 140, 2771–2780 Chen, H. et al. (2000) The cell biology of thrombospondin-1. Matrix. Biol. 19, 597–614 Chandrasekaran, L. et al. (2000) Cell contactdependent activation of α3β1 integrin modulates endothelial cell responses to thrombospondin-1. Mol. Biol. Cell. 11, 2885–2900 Jimenez, B. et al. (2000) Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nat. Med. 6, 41–48 Chen, H. et al. (1996) Metabolism of thrombospondin 2. Binding and degradation by 3T3 cells and glycosaminoglycan-variant Chinese hamster ovary cells. J. Biol. Chem. 271, 15993–15999 Adams, J. and Tucker, R. (2000) The thrombospondin type 1 repeat (TSR) superfamily: diverse proteins with related roles in neuronal development. Dev. Dyn. 218, 280–289
13 Hynes, R.O. and Zhao, Q. (2000) The evolution of cell adhesion. J. Cell. Biol. 150, F89–96 14 Lahav, J. (1993) The functions of thrombospondin and its involvement in physiology and pathophysiology. Biochim. Biophys. Acta 1182, 1–14 15 DiPietro, L.A. (1997) Thrombospondin as a regulator of angiogenesis. Exs 79, 295–314 16 Rastinejad, F. et al. (1989) Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell 56, 345–355 17 Good, D.J. et al. (1990) A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc. Natl. Acad. Sci. U. S. A. 87, 6624–6628 18 Volpert, O.V. et al. (1995) Inhibition of angiogenesis by thrombospondin-2. Biochem. Biophys. Res. Commun. 217, 326–332 19 Tolsma, S.S. et al. (1993) Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity. J. Cell. Biol. 122, 497–511
Review
20 Taraboletti, G. et al. (1990) Platelet thrombospondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor. J. Cell. Biol. 111, 765–772 21 Murphy-Ullrich, J.E. and Poczatek, M. (2000) Activation of latent TGF-β by thrombospondin-1: mechanisms and physiology. Cytokine Growth Factor Rev. 11, 59–69 22 Nor, J.E. et al. (2000) Thrombospondin-1 induces endothelial cell apoptosis and inhibits angiogenesis by activating the caspase death pathway. J. Vasc. Res. 37, 209–218 23 Taraboletti, G. et al. (2000) The heparin binding 25 kDa fragment of thrombospondin-1 promotes angiogenesis and modulates gelatinase and TIMP-2 production in endothelial cells. FASEB J. 14, 1674–1676 24 Lawler, J. et al. (1998) Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia. J. Clin. Invest. 101, 982–992 25 Kyriakides, T.R. et al. (1998) Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis. J. Cell Biol. 140, 419–430 26 Shweiki, D. et al. (1992) Vascular endothelial growth factor induced by hypoxia may mediate hypoxiainitiated angiogenesis. Nature 359, 843–845 27 Phelan, M.W. et al. (1998) Hypoxia increases thrombospondin-1 transcript and protein in cultured endothelial cells. J. Lab. Clin. Med. 132, 519–529 28 Laderoute, K.R. et al. (2000) Opposing effects of hypoxia on expression of the angiogenic inhibitor thrombospondin 1 and the angiogenic inducer vascular endothelial growth factor. Clin. Cancer Res. 6, 2941–2950 29 Tenan, M. et al. (2000) Thrombospondin-1 is downregulated by anoxia and suppresses tumorigenicity of human glioblastoma cells. J. Exp. Med. 191, 1789–1798 30 Dameron, K.M. et al. (1994) Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science 265, 1582–1584 31 Volpert, O.V. et al. (1997) Sequential development of an angiogenic phenotype by human fibroblasts progressing to tumorigenicity. Oncogene 14, 1495–1502 32 Van Meir, E.G. et al. (1994) Release of an inhibitor of angiogenesis upon induction of wild type p53 expression in glioblastoma cells. Nat. Genet. 8, 171–176 33 Grossfeld, G.D. et al. (1997) Thrombospondin-1 expression in bladder cancer: association with p53 alterations, tumor angiogenesis, and tumor progression. J. Natl. Cancer Inst. 89, 219–227 34 Kawahara, N. et al. (1998) Enhanced expression of thrombospondin-1 and hypovascularity in human cholangiocarcinoma. Hepatology 28, 1512–1517 35 Fontanini, G. et al. (1999) Thrombospondins I and II messenger RNA expression in lung carcinoma: relationship with p53 alterations, angiogenic growth factors, and vascular density. Clin. Cancer Res. 5, 155–161 36 Dejong, V. et al. (1999) The Wilms’ tumor gene product represses the transcription of thrombospondin 1 in response to overexpression of c-Jun. Oncogene 18, 3143–3151 37 Mettouchi, A. et al. (1994) SPARC and thrombospondin genes are repressed by the c-jun oncogene in rat embryo fibroblasts. EMBO J. 13, 5668–5678 http://tmm.trends.com
TRENDS in Molecular Medicine Vol.7 No.9 September 2001
38 Sheibani, N. and Frazier, W.A. (1996) Repression of thrombospondin-1 expression, a natural inhibitor of angiogenesis, in polyoma middle T transformed NIH3T3 cells. Cancer Lett. 107, 45–52 39 Slack, J.L. and Bornstein, P. (1994) Transformation by v-src causes transient induction followed by repression of mouse thrombospondin-1. Cell Growth Differ. 5, 1373–1380 40 Tikhonenko, A.T. et al. (1996) Viral Myc oncoproteins in infected fibroblasts down-modulate thrombospondin-1, a possible tumor suppressor gene. J. Biol. Chem. 271, 30741–30747 41 Zabrenetzky, V. et al. (1994) Expression of the extracellular matrix molecule thrombospondin inversely correlates with malignant progression in melanoma, lung and breast carcinoma cell lines. Int. J. Cancer 59, 191–195 42 Weidner, N. and Folkman, J. (1996) Tumoral vascularity as a prognostic factor in cancer. Important Adv. Oncol. 167–190 43 Clezardin, P. et al. (1993) Expression of thrombospondin (TSP1) and its receptors (CD36 and CD51) in normal, hyperplastic, and neoplastic human breast. Cancer Res. 53, 1421–1430 44 Oshiba, G. et al. (1999) Stromal thrombospondin-1 expression is correlated with progression of esophageal squamous cell carcinomas. Anticancer Res. 19, 4375–4378 45 Ohtani, Y. et al. (1999) Stromal expression of thrombospondin-1 is correlated with growth and metastasis of human gallbladder carcinoma. Int. J. Oncol. 15, 453–457 46 Bunone, G. et al. (1999) Expression of angiogenesis stimulators and inhibitors in human thyroid tumors and correlation with clinical pathological features. Am. J. Pathol. 155, 1967–1976 47 Bertin, N. et al. (1997) Thrombospondin-1 and -2 messenger RNA expression in normal, benign, and neoplastic human breast tissues: correlation with prognostic factors, tumor angiogenesis, and fibroblastic desmoplasia. Cancer Res. 57, 396–399 48 Pratt, D.A. et al. (1989) Thrombospondin in malignant and non-malignant breast tissue. Eur. J. Cancer Clin. Oncol. 25, 343–350 49 Ohta, Y. et al. (1999) Thrombospondin-1 expression and clinical implications in malignant pleural mesothelioma. Cancer 85, 2570–2576 50 Yoshida, Y. et al. (1999) Expression of angiostatic factors in colorectal cancer. Int. J. Oncol. 15, 1221–1225 51 De Fraipont, F. et al. (2000) Expression of the angiogenesis markers vascular endothelial growth factor, thrombospondin-1 and platelet-derived endothelial growth factor in human sporadic adrenocortical tumors. Correlation with genotypic alterations. J. Clin. Endocrinol. Metab. 85, 4734–4741 52 Oshika, Y. et al. (1998) Thrombospondin 2 gene expression is correlated with decreased vascularity in non-small cell lung cancer. Clin. Cancer Res. 4, 1785–1788 53 Hsu, S.C. et al. (1996) Inhibition of angiogenesis in human glioblastomas by chromosome 10 induction of thrombospondin-1. Cancer Res. 56, 5684–5691 54 Kazuno, M. et al. (1999) Thrombospondin-2 (TSP2) expression is inversely correlated with vascularity in glioma. Eur. J. Cancer 35, 502–506 55 Tokunaga, T. et al. (1999) Thrombospondin 2 expression is correlated with inhibition of angiogenesis and metastasis of colon cancer. Br. J. Cancer 79, 354–359 56 Souchelnitskiy, S. et al. (1995) Thrombospondins selectively activate one of the two latent forms of
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
407
transforming growth factor-β present in adrenocortical cell-conditioned medium. Endocrinology 136, 5118–5126 Yehualaeshet, T. et al. (1999) Activation of rat alveolar macrophage-derived latent transforming growth factor β-1 by plasmin requires interaction with thrombospondin-1 and its cell surface receptor, CD36. Am. J. Pathol. 155, 841–851 Crawford, S.E. et al. (1998) Thrombospondin-1 is a major activator of TGF-β1 in vivo. Cell 93, 1159–1170 Abdelouahed, M. et al. (2000) Activation of platelet transforming growth factor-β 1 in the absence of thrombospondin-1. J. Biol. Chem. 275, 17933–17936 Folkman, J. and D’Amore, P.A. (1996) Blood vessel formation: what is its molecular basis? Cell 87, 1153–1155 Andreasen, P.A. et al. (2000) The plasminogen activation system in tumor growth, invasion, and metastasis. Cell. Mol. Life Sci. 57, 25–40 Tuszynski, G.P. et al. (1992) Thrombospondin levels in patients with malignancy. Thromb. Haemost. 67, 607–611 Nathan, F.E. et al. (1994) Plasma thrombospondin levels in patients with gynecologic malignancies. Cancer 73, 2853–2858 Yamashita, Y. et al. (1998) Plasma thrombospondin levels in patients with colorectal carcinoma. Cancer 82, 632–638 Molema, G. et al. (1998) Tumor vasculature targeted therapies: getting the players organized. Biochem. Pharmacol. 55, 1939–1945 Weinstat-Saslow, D.L. et al. (1994) Transfection of thrombospondin 1 complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis. Cancer Res. 54, 6504–6511 Volpert, O.V. et al. (1998) A human fibrosarcoma inhibits systemic angiogenesis and the growth of experimental metastases via thrombospondin-1. Proc. Natl. Acad. Sci. U. S. A. 95, 6343–6348 Bleuel, K. et al. (1999) Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization. Proc. Natl. Acad. Sci. U. S. A. 96, 2065–2070 Streit, M. et al. (1999) Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas. Am. J. Pathol. 155, 441–452 Streit, M. et al. (1999) Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis. Proc. Natl. Acad. Sci. U. S. A. 96, 14888–14893 Bogdanov, A., Jr et al. (1999) Treatment of experimental brain tumors with thrombospondin-1 derived peptides: an in vivo imaging study. Neoplasia 1, 438–445 Castle, V. et al. (1991) Antisense-mediated reduction in thrombospondin reverses the malignant phenotype of a human squamous carcinoma. J. Clin. Invest. 87, 1883–1888 Wang, T.N. et al. (1996) Inhibition of breast cancer progression by an antibody to a thrombospondin-1 receptor. Surgery 120, 449–454 Maeda, K. et al. (2000) Expression of vascular endothelial growth factor and thrombospondin-1 in colorectal carcinoma. Int. J. Mol. Med. 5, 373–378 Nicosia, R.F. and Tuszynski, G.P. (1994) Matrix-bound thrombospondin promotes angiogenesis in vitro. J. Cell Biol. 124, 183–193
TRENDS in Molecular Medicine Vol.7 No.9 September 2001
401
Thrombospondins and tumor angiogenesis Florence de Fraipont, Ainsley C. Nicholson, Jean-Jacques Feige and Erwin G. Van Meir The thrombospondins (TSPs) are a family of five secreted proteins that are widely distributed in the extracellular matrix of numerous tissues. TSPs are multimodular and each domain specifies a distinct biological function through interaction with a specific receptor. TSP1 and TSP2 have anti-angiogenic activity, which, at least for TSP1, involves interaction with the microvascular endothelial cell receptor CD36. Expression of TSP1 and TSP2 is modulated by hypoxia and by oncogenes. In several tumors (thyroid, colon, bladder carcinomas), TSP1 expression is inversely correlated with tumor grade and survival rate, whereas in others (e.g. breast carcinomas), it is correlated with the stromal response and is of little prognostic value. Recent studies suggest that TSPs or TSP-derived peptides retaining biological activity could be developed into promising new therapeutic strategies for the anti-angiogenic treatment of solid tumors.
Florence de Fraipont Jean-Jacques Feige INSERM EMI 0105, Dept of Molecular and Structural Biology, Commissariat à l’Energie Atomique, Grenoble, France. Ainsley C. Nicholson Erwin G. Van Meir* Laboratory of Molecular Neuro-Oncology, Neurosurgery Dept and Winship Cancer Institute, Emory University, Atlanta, GA, 30322, USA. *e-mail: [email protected]
Platelet thrombospondin (now named TSP1) is the canonical member of a family of structurally related proteins with five distinct members, and was named to reflect its initial purification from thrombin-activated platelet releasates. TSP1 was later shown to be secreted from a wide variety of epithelial and mesenchymal cells. All five TSP family members are multimeric and multimodular heparinand calcium-binding proteins, but two subfamilies can be distinguished1–3. TSP1 and TSP2 have a similar structural organization, as shown in Fig. 1. Each subunit of these trimeric molecules is composed of an N-terminal heparin-binding domain, a linker domain enclosing the two cysteine residues implicated in trimerization, a procollagen-homology domain, three properdin-like type I repeats, three epidermal growth factor (EGF)-like type II repeats, seven calcium-binding type III repeats and a globular C-terminal domain. The remaining TSP proteins, TSP3, TSP4 and TSP5/COMP, resemble one another but differ from TSP1 and TSP2 in that they are pentameric, and their subunits possess four instead of three type II repeats and lack the procollagenhomology domain and the three type I repeats (Fig. 1). Regulation and expression patterns of the TSP family members differ. During murine development, TSP1 is expressed in liver, kidney, and gut, and TSP2 is expressed primarily in connective tissues4. In adult tissues, TSP1 and TSP2 are expressed in a variety of organs and have overlapping but distinct expression patterns5–7. Other members are particularly abundant in cartilage and bone tissues. A variety of cellular receptors that recognize specific peptide domains of TSP1 have been identified (Fig. 2). They appear to mediate a variety of biological functions8. CD47/IAP (integrin-associated protein) binds to the C-terminal domain, integrins αvβ3 and http://tmm.trends.com
αIIbβ3 bind to RGD motifs in the last type III repeats, and binding of the scavenger receptor LRP (LDL receptor-related protein) is dependent upon association of TSP1 with heparan sulfate proteoglycans (HSPGs). Recently, a sequence in the N-terminal domain was also found to bind integrin α3β1 on tumor cells and aortic endothelial cells9. Most interestingly, discrete repetitive sequences (comprising CSVTCG and adjacent sequences) present in the second and third type I repeats mediate the binding of TSP1 to CD36 on endothelial cells, resulting in inhibition of angiogenesis3,10. Although all these sequences are well conserved in TSP2, only the HSPG binding and the HSPG-mediated interaction with LRP has been clearly demonstrated using the full-length TSP2 protein11. Almost 70 proteins in the human genome also possess type I repeat motifs (Ainsley C. Nicholson and Erwin G. Van Meir, unpublished). A subset of these affect angiogenesis (Fig. 3). Brain angiogenesis inhibitor (BAI)-1 and ADAMTS-1 and -8 – two members of the ADAMTS (a disintegrin and metalloproteinase with TSP motifs) family – inhibit angiogenesis, whereas connective tissue growth factor (CTGF), which possesses a single type I repeat, stimulates angiogenesis12. By contrast, several proteins containing type I repeats have no angiogenic effects. These include complement component proteins from the alternate immune response pathway (including C6, C7, C8, C9), properdin, the neurotrophic factors F-spondin, SCO-spondin, semaphorins 5A and 5B and several other ADAMTS proteins12. Most proteins containing TSP-type I repeats have an affinity for heparin, but the elements within type I repeats that might confer pro- or anti-angiogenic activity are still being determined. It should be noted that, despite the absence of TSPs from the genomes of non-vascularized invertebrate metazoans such as Caenorhabditis elegans or Drosophila melanogaster, some proteins of these species do contain type I repeat motifs, indicating that this functional motif arose early and has been preserved through evolution13. TSP1 and TSP2 are anti-angiogenic
Our current appreciation of the involvement of TSPs in diverse biological processes extends far beyond the initial observations of their roles in platelet aggregation and coagulation2,14. TSP1 released by activated platelets participates in the formation and resolution of the fibrin clot, by binding to fibrin, plasminogen and urokinase through its N-terminal
1471-4914/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1471-4914(01)02102-5
Review
402
Fig. 1. Members of the thrombospondin (TSP) family have a multimodular structure. TSP1 and TSP2 are composed of an N-terminal heparin-binding domain (large green circle), a procollagen homology domain (dark-blue oval), three type I repeats (green circles), three epidermal growth factor (EGF)-like type II repeats (orange diamonds), seven calcium-binding type III repeats (pale blue rectangles) and a C-terminal globular domain (blue circle). By contrast, TSP3, TSP4 and TSP5/COMP contain distinct N-terminal domains (light-blue, red, purple circles), four EGFlike repeats, seven type III repeats and a similar C-terminal globular domain. ‘S’ indicates the position of the cysteine residues involved in multimerization. Sequence homology increases from the N- to the C-termini of these proteins.
TRENDS in Molecular Medicine Vol.7 No.9 September 2001
TSP1 S
COOH
NH2 S TSP2 S S TSP3 S S TSP4 S S TSP5/COMP S S
TRENDS in Molecular Medicine
heparin-binding domain. TSP1 also participates in the formation of molecular bridges between platelets and inflammatory leukocytes through interactions with cell-surface receptors such as CD36 or the αvβ3 and αIIbβ3 integrins. The regulatory role of TSPs on endothelial cell adhesion, migration and proliferation has been intensely studied in vitro since the late 1980s15–18. Human platelet TSP1 specifically inhibits both the proliferation of endothelial cells from various tissue origins and their three-dimensional organization into capillary-like structures when grown in collagen gels. TSP1 was also found to inhibit neovascularization in vivo and endothelial cell migration toward fibroblast growth factor (FGF)-2 in vitro (ED50 ~1 nM)17,19. However, it should be noted that, at
Trimerization
Latent TGFβ activation RFK
S COOH
NH2 S WXXW CSVTCG
QNV α3β1
HSPG
Cell adhesion Hemostasis
CD36 Anti-angiogenesis cell adhesion
RGD
RFYVVM IRVVM
αvβ3
IAP/CD47 αIIbβ3 Cell adhesion Cell adhesion
LRP gp330 Internalization / degradation TRENDS in Molecular Medicine
Fig. 2. Multiple receptors recognize thrombospondin (TSP)1. Almost every domain of the TSP1 molecule binds to a specific receptor. The peptide sequences indicated below each module are specifically recognized by the receptors indicated in oval boxes. Two distinct domains of the TSP1 molecule bind heparan sulfate proteoglycans (HSPG), and this binding appears to be a prerequisite for binding to the scavenger receptor LRP. The biological function of each receptor is indicated below each box. Abbreviations: IAP, integrin-associated protein; LRP, low-density lipoprotein receptor-related protein; TGFβ, transforming growth factor β.
http://tmm.trends.com
higher concentrations (20–100 nM), TSP1 promotes endothelial cell migration19,20. Using proteolytic fragments and synthetic peptides, the angio-inhibitory activity of TSP1 has been mapped to the procollagen homology domain and to the type I repeats19. Two separate non-overlapping peptides [one of them containing a latent transforming growth factor (TGF)β-activating sequence21] derived from the second and third type I repeats displayed anti-angiogenic activity. However, the anti-angiogenic activity of TSP1 appears to function independently from its activation of latent TGFβ (Ref. 21). TSP2 also contains type I repeats, and recombinant mouse TSP2 and natural bovine TSP2 have been shown by in vitro assays to inhibit angiogenesis to a similar degree as TSP1, whereas TSP5/COMP, a pentameric member of the TSP family lacking type I repeats, is inactive18. The anti-angiogenic action of the CSVTCGcontaining type I repeats in TSP1 is mediated by the interaction with the membrane receptor CD36 present on the surface of blood capillaries. As shown in Figure 4, this leads to the sequential activation of cytoplasmic tyrosine kinase p59fyn, caspase-3-like proteases and p38 mitogen-activated protein (MAP) kinase and results in endothelial cell apoptosis10. Endothelial cells exposed to TSP1 display increased levels of Bax expression, decreased levels of Bcl-2 expression, and process caspase-3 into smaller pro-apoptotic forms22. However, other investigators found a pro-angiogenic activity for TSP1. Nicosia and Tuszynski75 observed that the presence of TSP1 in serum-free cultures of aortic ring explants in a collagen gel promoted the number and length of microvessels outgrowing the explants. As observed with cell migration, this effect was observed at high TSP1 concentrations (ED50 ~20–40 nM) and might result from the interaction of TSP1 domains other than those shown to have anti-angiogenic activity with a distinct receptor. In accordance with this hypothesis, Taraboletti et al.23 recently reported that the N terminal heparin-binding domain of TSP1 is angiogenic in the rabbit cornea assay, whereas the 140-kDa fragment lacking this domain appears to inhibit the FGF2-driven angiogenic response. The activity of the intact TSP proteins and their derived peptides might be very dependent on their conformation. It was recently shown that engagement of the α3β1 integrin by a soluble TSP-1 peptide inhibits endothelial cell proliferation and angiogenesis, whereas the same peptide stimulates endothelial cell proliferation when immobilized9. The role of TSP1 and 2 in embryonic angiogenesis has been addressed in several animal studies. During mouse embryonic development, TSP1 mRNA is detected in capillaries of day 16–18 embryos4. However, disruption of the TSP1 gene in mice does not appear to dramatically alter the normal embryonic development of the vasculature, as these animals are viable and appear to develop normally24. TSP2 is expressed in capillaries and large vessels
Review
Fig. 3. Several proteins regulating angiogenesis possess thrombospondin (TSP)-like type I repeats. TSP type I repeats (small green circles), which govern the angiostatic activity of thrombospondin-1, are found in several proteins that affect angiogenesis. A disintegrin and metalloproteinase with TSP repeats (ADAMTS)-1 and -8 and brain angiogenesis inhibitor (BAI)-1 are angiostatic, whereas connective tissue growth factor (CTGF) promotes angiogenesis. Other known proteins containing type I repeats have not been shown to affect angiogenesis.
TRENDS in Molecular Medicine Vol.7 No.9 September 2001
S
TSP1
403
TSP1
S
Endothelial cell ADAMTS-1,8
CD36
P
p38
p59fyn
Nucleus
Apoptosis
Caspases (3-like)
P BAI1 Caspases (3-like)
pp38 Angiogenesis
CTGF TRENDS in Molecular Medicine TRENDS in Molecular Medicine
from day 10 post-conception continuously through parturition on day 18 (Ref. 4). The phenotype of TSP2-knockout mice is more dramatic than that of TSP1 knockouts25. Although viable, these mice develop alterations in the tensile strength of skin and tendons, an abnormal bleeding time and an increased density of blood vessels in many tissues. This last feature is a clear indicator of the importance of the role of TSP2 in the regulation of organ vascularity. Expression and regulation of TSP1 and TSP2 Regulation by hypoxia
Hypoxia strongly favors angiogenesis through induction of vascular endothelial growth factor (VEGF) expression and improved VEGF mRNA stability26. It was therefore of interest to determine whether hypoxia also affects TSP1 expression. In endothelial cells (HUVEC or HMVEC), hypoxia was reported to induce TSP1 gene and protein expression by post-transcriptional stabilization of the TSP1 mRNA27. By contrast, hypoxia strongly decreases TSP1 mRNA levels in both p53+/+ and p53−/− human and rodent fibroblasts and in several human glioblastoma tumor cell lines28,29. No significant effect was observed in human cervical epithelial cells28, and TSP1 expression was unaffected by hypoxia in bovine adrenocortical cells (but TSP2 mRNA and protein levels were downregulated) (M. Keramidas and J.J. Feige, personal communication). These observations suggest that the regulation of TSP1 and TSP2 expression by hypoxia could depend on tissue type, cell transformation, and experimental conditions. Although downregulation of TSP1 by tumor cells appears to promote angiogenesis, upregulation by endothelial cells could be part of a negative feedback loop, although this remains to be established. Furthermore, it is unclear whether downregulation by hypoxia is a specific regulatory mechanism or the result of decreased metabolic activity under hypoxia. Regulation by oncogenes and tumor-suppressor genes
The initial observation that TSP1 possesses anti-angiogenic activity, and the later discovery that genetic alteration of certain tumor suppressor genes http://tmm.trends.com
Fig. 4. Thrombospondin (TSP)-1 induces endothelial cell apoptosis. TSP1 binds through its type I repeats to CD36 receptors present at the surface of microvascular endothelial cells and activates the cytoplasmic protein tyrosine-kinase p59fyn. This kinase in turn activates caspase-3-like proteases, which activate p38 mitogen-activated protein kinase. Translocation of the phosphorylated form of p38 mitogen-activated protein kinase into the nucleus induces expression of caspase-3 resulting in endothelial cell apoptosis. Adapted from Ref. 10.
correlates with simultaneous loss of TSP1 expression and acquisition of an angiogenic phenotype by tumor cells16,17 suggests an exciting new paradigm for tumor progression. Among tumor suppressor loci affecting TSP1 expression, TP53 is the best characterized. The TP53 tumor suppressor gene is mutated in over 50% of human cancers. Spontaneous loss, after several passages in culture, of the wild-type (wt) TP53 allele from human Li-Fraumeni fibroblasts with a TP53 wt/mutant genotype was accompanied by a simultaneous decrease in the secretion of TSP1, whereas TSP2 expression remained stable. Reintroduction of the wt TP53 gene into these fibroblasts restored TSP1 mRNA levels30,31, confirming that the reduction in TSP1 expression was causally related to the loss of p53 activity. Such regulatory mechanisms might be tissue-specific, as modulation of exogenous p53 did not modify cellular TSP1 levels in the p53-null human glioma cell line LN-Z308 (Refs 29,32). Analysis of patients with invasive transitional cell carcinoma of the bladder determined that reduced TSP1 expression correlates with the presence of p53 alterations in tumors and with increased recurrence rates, increased microvessel density, and decreased overall survival33. Such a clear-cut association might not be a general feature for all tumor types, as analysis of certain cancers, including lung cancer, cholangiocarcinoma, and glioblastoma, found no association between the presence of p53 alterations and TSP1 mRNA expression29,34,35. Oncogenes were also found to regulate TSP1 expression in the Li-Fraumeni model described above. Introduction of activated ras resulted in cell transformation and a further decrease in TSP1 mRNA levels31. Inhibition of basal TSP1 expression by various oncogenes or oncogenic signals including not only ras but also v-src, v-myc, polyoma middle T antigen and overexpressed c-jun has been reported36–41. However,
404
Review
TRENDS in Molecular Medicine Vol.7 No.9 September 2001
this is not a universal feature of oncogenes, as SV40 large T or BPV1 did not inhibit TSP1 expression37. Thus, research has demonstrated that both oncogene activation and loss of tumor-suppressor gene expression are directly or indirectly associated with a decrease in TSP1 expression. This could increase the likelihood of the tumor gaining the capacity to induce blood vessel formation, thereby making the ‘angiogenic switch’ and progressing towards greater malignancy. Expression of TSP1 and TSP2 in human cancers
Several studies have demonstrated that the extent of tumor angiogenesis is related to clinical outcome42. As TSP1 and TSP2 inhibit angiogenesis, and TSP1 expression can be downregulated by oncogene activation or tumor suppressor loss, it has been hypothesized that their expression could be inversely correlated with tumor progression, recurrence or metastatic potential (see Table 1). The results of several studies aimed at determining TSP1 levels in different tumor types, by Northern-blot, RT-PCR or immunohistochemistry, are reported in Table 1. In evaluating these results, it is important to differentiate between expression of TSP1 and/or TSP2 by tumor cells, and expression in stromal tissues that surround a tumor. This can only be achieved by immunohistochemical or in situ hybridization analyses. In several distinct tumors including breast, cholangial, esophageal, gallbladder, thyroid and bladder carcinomas, TSP1 was present in the stroma and weakly expressed or absent in the tumor cells33,34,43–46. Many other studies using RT-PCR or biochemical methods to detect TSPs did not address the question of stromal versus tumoral distribution but rather determined an overall content in the analyzed tissue specimens. However, from the results of such studies, one can differentiate several tumors in which TSP1 or TSP2 expression is inversely correlated to tumor grade and others in which TSP1 or TSP2 expression is higher in carcinomas than in benign tumors or normal tissue. Human tumors in which increased TSP1 or TSP2 expression is associated with malignancy
In breast tumors and cholangiocarcinomas, the stromal expression of TSP1 was much stronger in tumors than in normal tissue34,43,47,48. Similarly, in colorectal carcinomas and pleural mesothelioma, the level of TSP1 expression was higher in tumors than in normal tissue49,50. In all of these tumors, TSP1 levels were of little prognostic value. By contrast, in esophageal and gallbladder carcinomas, TSP1 expression appeared to be directly correlated with the occurrence of metastases44,45. Human tumors in which decreased TSP expression is associated with malignancy
In human adrenocortical carcinomas, non-small cell lung carcinomas, gliomas and thyroid carcinomas, http://tmm.trends.com
TSP levels were lower in malignant tumors than in adenomas29,46,51–54. Several studies showed that TSP1 expression is inversely correlated with tumor grade and survival rate in thyroid, colon and bladder carcinomas33,46,55. In non-small cell lung carcinoma, only TSP2 expression (not TSP1) appeared as a significant prognostic marker52. Similarly, increased TSP1 expression by tumor cells was found to correlate with reduced metastatic potential in experimental tumors formed from melanoma, lung, and breast carcinoma cell lines41. One possible explanation for the different effects of TSP1 expression in various tumor types relates to its interaction with TGFβ. TSP1 binds to the latent precursor of TGFβ via the RFK sequence (located between the first and the second type I repeats) and the WXXW sequences. This favors the release of active TGFβ, either directly21 or indirectly56,57. The importance of this interaction is suggested by the similarity of histological abnormalities in nine organ systems between TGFβ null and TSP1 null mice58, although other physiological latent TGFβ activation mechanisms exist59. TGFβ triggers apoptosis in several tumor-cell types, and might also contribute to tumor growth by stabilizing the tumor vasculature60. In cancer formation, the release of active TGFβ can result in upregulation of uPA (urokinase plasminogen activator), uPA-receptor and PAI-1 (plasminogen activator inhibitor-1), three components of the proteolytic system that promotes malignant invasiveness61. This promotion of malignancy by interaction with TGFβ acts in opposition to the anti-tumorigenic/anti-angiogenic activities of TSP1. The overall effect of TSP1 overexpression on tumor cells could, therefore, reflect the balance between anti-angiogenic forces versus invasive forces. For a specific tumor, whether TSP overexpression promotes or inhibits malignancy could be at least partially dependent on the concentration of TGFβ at the tumor site. Plasma levels of TSP1
Plasma TSP1 levels in patients with metastatic breast, lung or gastrointestinal carcinomas were found to be two- to three-fold higher than those of healthy volunteers or patients with non-metastatic disease62. A direct correlation was established between plasma TSP1 level and the stage of disease in patients with gynecologic or colorectal malignancies63,64. As the origin of plasma TSP1 (platelet clotting, endothelial cells or tumor cells) has not been completely characterized, and the function of circulating TSP1 in cancer patients is unknown, it is difficult to determine whether high plasma TSP1 concentrations are beneficial in reducing growth of distant metastases for patients with certain tumor types, or produce a detrimental increase in metastatic invasiveness. It is also not clear whether plasma TSP1 levels can be used as a prognostic factor.
Review
TRENDS in Molecular Medicine Vol.7 No.9 September 2001
405
Table 1. Correlations between thrombospondin (TSP) levels and clinical features in different human cancers Tumor type
Cohort Method size
Results
Refs
Bladder carcinoma
163
Immunohistochemistry
TSP1 expression is inversely correlated with disease recurrence and overall survival
33
Breast carcinoma
101
Radioimmunoassay
High levels of TSP1 found in carcinomas; absence of correlation with clinical parameters
48
25
Immunohistochemistry and Weak TSP1 staining in normal tissue; strong staining in stroma around in situ hybridization tumor cells
43
3
Northern blot
TSP1 is inversely correlated with malignant progression
41
33
RT-PCR
TSP1 and TSP2 expressions are higher in invasive carcinomas than in normal or benign tissue; no correlation with clinical parameters or vascularity
47
Colorectal carcinoma 61
RT-PCR
TSP2 expression is inversely correlated with the occurrence of distant metastases; no correlation of TSP1 expression with clinical parameters
55
62
RT-PCR
TSP2 is more frequently expressed in tumoral than in extraneoplastic colon mucosa; TSP1 expression not significantly different
50
100
Immunohistochemistry
TSP1 expression is inversely correlated with good prognosis
74
Cholangiocarcinoma 11
Northern blot and immunohistochemistry
TSP1 expression is higher in tumoral than in surrounding healthy tissue; no correlation with clinical parameters
34
Adrenocortical carcinoma
43
ELISA
Lower TSP1 concentrations in carcinomas and transitional tumors than in adenomas
51
Esophageal squamous cell carcinoma
54
Immunohistochemistry
Higher TSP1 expression in tumoral than in healthy tissue; TSP1 immunoreactivity detected in the stroma, infrequently in the cancer cells; higher incidence of lymph node metastasis in the TSP1-positive cases
44
Gallbladder carcinoma
53
RT-PCR and immunohistochemistry
TSP1 immunoreactivity detected in the stroma, infrequently in the cancer cells; TSP1 expression is correlated with lymph node metastasis
45
Glioma
17
Immunohistochemistry
Strong TSP1 staining in normal brain, in low-grade astrocytomas and in anaplastic astrocytomas; weak or no staining in high-grade glioblastomas
53
37
RT-PCR
TSP2 gene expression is inversely associated with histological grade; no correlation of TSP1 expression with clinical parameters
54
10
Immunohistochemistry
Of recurring gliomas in which TSP1 expression was found, 4/5 had decreased TSP1 expression in higher-grade gliomas
29
Non-small cell lung carcinoma
78
RT-PCR
Decreased TSP2 expression and inverse correlation with good prognosis; no correlation of TSP1 expression with clinical parameters
52
Malignant pleural mesothelioma
78
RT-PCR
TSP1 is overexpressed but its expression has little prognostic value
49
Thyroid carcinoma
84
RT-PCR and immunohistochemistry
Downregulation of TSP1 expression in aggressive carcinomas
46
Anti-angiogenic thrombospondins in cancer therapy
Since the discovery of the role of angiogenesis in the pathogenesis of tumor growth and metastasis, new cancer treatment strategies exploiting selective inhibition of tumor neovascularization have been explored65. In this context, TSP1 and TSP2 have been tested as anti-tumor agents in several xenograft models. Animal models in which TSP1 or TSP2 decreases tumor growth
Transfection of glioblastoma, fibrosarcoma, breast carcinoma and cutaneous carcinoma cells with a plasmid expressing the TSP1 protein results in reduced tumor growth in immunocompromised nude (nu/nu) mice29,66–69. Similar results were observed when squamous epithelial carcinoma cells were transfected with a TSP2 expression plasmid70. Moreover, in this model, TSP2 was significantly more active than TSP1 and had synergistic antitumoral http://tmm.trends.com
effects with TSP1 that resulted in complete suppression of tumor development70. In all these models, inhibition of tumor growth was generally accompanied by a decrease in tumor vascularization confirming the hypothesis that angiostatic factors could be used to inhibit tumor growth. Impressive inhibition of tumor growth with potentially angiostatic peptides from TSP1 was observed in intracerebral rat glioma models (C6, 9L)71. Surprisingly, a difference in vascular density could not be detected when in vivo imaging was used71, perhaps because of the limited resolution of this technique (250–500 µm range), suggesting that the methods chosen for precise visualization of the capillary networks are critical. Systemic release of high levels of TSP1 can also inhibit angiogenesis. Expression of high levels of TSP1 by a human fibrosarcoma line implanted subcutaneously in nude mice reduced growth of experimental melanoma metastases in the lung of the
406
Review
TRENDS in Molecular Medicine Vol.7 No.9 September 2001
same animals. Apparently, systemic TSP1 released from the fibrosarcoma prevented the metastases from establishing vascularization, thereby reducing their growth. This effect could be recapitulated by direct injection of purified TSP1 protein67. Animal models in which TSP1 augments tumor growth
Other animal models, however, cast doubt on the anti-tumorigenic potential of TSP1. Although overexpression of TSP1 by breast cancer cells resulted in smaller tumors than controls, overexpression in the same cell of a TSP1 protein fragment lacking the last 98 C-terminal residues resulted in tumors that grew larger than control tumors. This was surprising, because the truncated TSP1 had retained previously identified angiostatic domains66. Similarly, cancer progression was inhibited by neutralization of TSP1 expression in squamous carcinoma by anti-sense mRNA (Ref. 72) and in breast tumors by a polyclonal anti-TSP1 receptor antibody73. These observed differences in effect of TSP1 expression on tumor growth might be more fully explained when we have a better understanding of the specific functions of TSP1 in various cell types. Clinical trials with TSP1 and TSP2 Acknowledgements E.G.V.M. was supported by grants from the NIH (NS 41403 and CA86335), the Swiss National Science Foundation (4037-044729), and the University Research Committee of Emory University. A.C.N. was supported by NIH training grant T32 NS07480. F.d.F. and J.J.F. were supported by INSERM (EMI 0105), CEA and grants from the Ligue Nationale contre le Cancer (Comités de l’Isère et de la Drôme) and the GEFLUC.
Because of their large size (> 450 kDa), TSP1 and TSP2 have not been used in clinical trials. Peptides derived from TSP1 type I repeats, the region which is critical for inducing endothelial cell apoptosis after engaging the CD36 receptor10, might have clinical applications. Methods for stabilizing these peptides in vivo and delivering them to tumor sites are currently being researched, and will be essential in the development of cancer treatments based on TSP1 or TSP2. Conclusion
Of the five members of the TSP family of proteins, only TSP1 and TSP2, which contain type I repeat motifs, display anti-angiogenic activity. The
References 1 Bornstein, P. and Sage, E.H. (1994) Thrombospondins. Methods Enzymol. 245, 62–85 2 Feige, J-J. (2000) Thrombospondins. In Encyclopedic Reference of Vascular Biology and Pathology (Bikfalvi, A., ed.), pp. 285–292, Springer 3 Lawler, J. (2000) The functions of thrombospondin-1 and -2. Curr. Opin. Cell. Biol. 12, 634–640 4 Iruela-Arispe, M.L. et al. (1993) Differential expression of thrombospondin 1, 2, and 3 during murine development. Dev. Dyn. 197, 40–56 5 Corless, C.L. et al. (1992) Colocalization of thrombospondin and syndecan during murine development. Dev. Dyn. 193, 346–358 6 Kyriakides, T.R. et al. (1998) The distribution of the matricellular protein thrombospondin 2 in tissues of embryonic and adult mice. J. Histochem. Cytochem. 46, 1007–1015 7 Danik, M. et al. (1999) Bovine thrombospondin-2: complete complementary deoxyribonucleic acid http://tmm.trends.com
8 9
10
11
12
regulation of TSP1 expression by tumor suppressors and oncogenes not only implies that the angiogenic switch during tumor formation is facilitated by loss of TSP1 expression, it also suggests possible uses for the anti-angiogenic properties of these proteins, or TSP-derived peptides retaining anti-angiogenic activity, in cancer therapy. Although the differing results of various studies correlating levels of TSP expression with tumor progression and clinical prognosis is perplexing at this time, they do indicate a complexity of interaction between TSPs and various cell types. These differences could be caused by as yet undiscovered receptors, or by varying responses within the different cell types to stimulation by TSPs. This could limit the usefulness of anti-tumor gene therapy using TSPs to certain tumor types. It should be noted, however, that in tumor types that do respond to TSP stimulation, the anti-tumorigenic response is dramatic. This certainly encourages continued research into the use of TSPs, particularly TSP1 and TSP2, as anti-cancer agents. Future investigations are expected to reveal the molecular basis for the differing effects of TSP1 on tumorigenesis in different tumor types, and to describe the molecular pathways for the regulation of TSP1 by multiple tumor suppressors and oncogenes. Research into the possibility of using TSP1 or TSP2 as prognostic markers for cancer patients could improve the precision of clinical predictions. Cancer therapies using TSPs might be developed, and it will be particularly interesting to learn if TSP type I repeat peptides or peptide mimetics can be utilized in clinical therapy and how they compare with other anti-angiogenic factors such as TNP-470, angiostatin, and endostatin. There are issues to resolve before the anti-angiogenic properties of TSP1 and TSP2 can be employed for the treatment of cancer, but substantial progress has already been made, and the intense research in this field promises many new discoveries.
sequence and immunolocalization in the external zones of the adrenal cortex. Endocrinology 140, 2771–2780 Chen, H. et al. (2000) The cell biology of thrombospondin-1. Matrix. Biol. 19, 597–614 Chandrasekaran, L. et al. (2000) Cell contactdependent activation of α3β1 integrin modulates endothelial cell responses to thrombospondin-1. Mol. Biol. Cell. 11, 2885–2900 Jimenez, B. et al. (2000) Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nat. Med. 6, 41–48 Chen, H. et al. (1996) Metabolism of thrombospondin 2. Binding and degradation by 3T3 cells and glycosaminoglycan-variant Chinese hamster ovary cells. J. Biol. Chem. 271, 15993–15999 Adams, J. and Tucker, R. (2000) The thrombospondin type 1 repeat (TSR) superfamily: diverse proteins with related roles in neuronal development. Dev. Dyn. 218, 280–289
13 Hynes, R.O. and Zhao, Q. (2000) The evolution of cell adhesion. J. Cell. Biol. 150, F89–96 14 Lahav, J. (1993) The functions of thrombospondin and its involvement in physiology and pathophysiology. Biochim. Biophys. Acta 1182, 1–14 15 DiPietro, L.A. (1997) Thrombospondin as a regulator of angiogenesis. Exs 79, 295–314 16 Rastinejad, F. et al. (1989) Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell 56, 345–355 17 Good, D.J. et al. (1990) A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc. Natl. Acad. Sci. U. S. A. 87, 6624–6628 18 Volpert, O.V. et al. (1995) Inhibition of angiogenesis by thrombospondin-2. Biochem. Biophys. Res. Commun. 217, 326–332 19 Tolsma, S.S. et al. (1993) Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity. J. Cell. Biol. 122, 497–511
Review
20 Taraboletti, G. et al. (1990) Platelet thrombospondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor. J. Cell. Biol. 111, 765–772 21 Murphy-Ullrich, J.E. and Poczatek, M. (2000) Activation of latent TGF-β by thrombospondin-1: mechanisms and physiology. Cytokine Growth Factor Rev. 11, 59–69 22 Nor, J.E. et al. (2000) Thrombospondin-1 induces endothelial cell apoptosis and inhibits angiogenesis by activating the caspase death pathway. J. Vasc. Res. 37, 209–218 23 Taraboletti, G. et al. (2000) The heparin binding 25 kDa fragment of thrombospondin-1 promotes angiogenesis and modulates gelatinase and TIMP-2 production in endothelial cells. FASEB J. 14, 1674–1676 24 Lawler, J. et al. (1998) Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia. J. Clin. Invest. 101, 982–992 25 Kyriakides, T.R. et al. (1998) Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis. J. Cell Biol. 140, 419–430 26 Shweiki, D. et al. (1992) Vascular endothelial growth factor induced by hypoxia may mediate hypoxiainitiated angiogenesis. Nature 359, 843–845 27 Phelan, M.W. et al. (1998) Hypoxia increases thrombospondin-1 transcript and protein in cultured endothelial cells. J. Lab. Clin. Med. 132, 519–529 28 Laderoute, K.R. et al. (2000) Opposing effects of hypoxia on expression of the angiogenic inhibitor thrombospondin 1 and the angiogenic inducer vascular endothelial growth factor. Clin. Cancer Res. 6, 2941–2950 29 Tenan, M. et al. (2000) Thrombospondin-1 is downregulated by anoxia and suppresses tumorigenicity of human glioblastoma cells. J. Exp. Med. 191, 1789–1798 30 Dameron, K.M. et al. (1994) Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science 265, 1582–1584 31 Volpert, O.V. et al. (1997) Sequential development of an angiogenic phenotype by human fibroblasts progressing to tumorigenicity. Oncogene 14, 1495–1502 32 Van Meir, E.G. et al. (1994) Release of an inhibitor of angiogenesis upon induction of wild type p53 expression in glioblastoma cells. Nat. Genet. 8, 171–176 33 Grossfeld, G.D. et al. (1997) Thrombospondin-1 expression in bladder cancer: association with p53 alterations, tumor angiogenesis, and tumor progression. J. Natl. Cancer Inst. 89, 219–227 34 Kawahara, N. et al. (1998) Enhanced expression of thrombospondin-1 and hypovascularity in human cholangiocarcinoma. Hepatology 28, 1512–1517 35 Fontanini, G. et al. (1999) Thrombospondins I and II messenger RNA expression in lung carcinoma: relationship with p53 alterations, angiogenic growth factors, and vascular density. Clin. Cancer Res. 5, 155–161 36 Dejong, V. et al. (1999) The Wilms’ tumor gene product represses the transcription of thrombospondin 1 in response to overexpression of c-Jun. Oncogene 18, 3143–3151 37 Mettouchi, A. et al. (1994) SPARC and thrombospondin genes are repressed by the c-jun oncogene in rat embryo fibroblasts. EMBO J. 13, 5668–5678 http://tmm.trends.com
TRENDS in Molecular Medicine Vol.7 No.9 September 2001
38 Sheibani, N. and Frazier, W.A. (1996) Repression of thrombospondin-1 expression, a natural inhibitor of angiogenesis, in polyoma middle T transformed NIH3T3 cells. Cancer Lett. 107, 45–52 39 Slack, J.L. and Bornstein, P. (1994) Transformation by v-src causes transient induction followed by repression of mouse thrombospondin-1. Cell Growth Differ. 5, 1373–1380 40 Tikhonenko, A.T. et al. (1996) Viral Myc oncoproteins in infected fibroblasts down-modulate thrombospondin-1, a possible tumor suppressor gene. J. Biol. Chem. 271, 30741–30747 41 Zabrenetzky, V. et al. (1994) Expression of the extracellular matrix molecule thrombospondin inversely correlates with malignant progression in melanoma, lung and breast carcinoma cell lines. Int. J. Cancer 59, 191–195 42 Weidner, N. and Folkman, J. (1996) Tumoral vascularity as a prognostic factor in cancer. Important Adv. Oncol. 167–190 43 Clezardin, P. et al. (1993) Expression of thrombospondin (TSP1) and its receptors (CD36 and CD51) in normal, hyperplastic, and neoplastic human breast. Cancer Res. 53, 1421–1430 44 Oshiba, G. et al. (1999) Stromal thrombospondin-1 expression is correlated with progression of esophageal squamous cell carcinomas. Anticancer Res. 19, 4375–4378 45 Ohtani, Y. et al. (1999) Stromal expression of thrombospondin-1 is correlated with growth and metastasis of human gallbladder carcinoma. Int. J. Oncol. 15, 453–457 46 Bunone, G. et al. (1999) Expression of angiogenesis stimulators and inhibitors in human thyroid tumors and correlation with clinical pathological features. Am. J. Pathol. 155, 1967–1976 47 Bertin, N. et al. (1997) Thrombospondin-1 and -2 messenger RNA expression in normal, benign, and neoplastic human breast tissues: correlation with prognostic factors, tumor angiogenesis, and fibroblastic desmoplasia. Cancer Res. 57, 396–399 48 Pratt, D.A. et al. (1989) Thrombospondin in malignant and non-malignant breast tissue. Eur. J. Cancer Clin. Oncol. 25, 343–350 49 Ohta, Y. et al. (1999) Thrombospondin-1 expression and clinical implications in malignant pleural mesothelioma. Cancer 85, 2570–2576 50 Yoshida, Y. et al. (1999) Expression of angiostatic factors in colorectal cancer. Int. J. Oncol. 15, 1221–1225 51 De Fraipont, F. et al. (2000) Expression of the angiogenesis markers vascular endothelial growth factor, thrombospondin-1 and platelet-derived endothelial growth factor in human sporadic adrenocortical tumors. Correlation with genotypic alterations. J. Clin. Endocrinol. Metab. 85, 4734–4741 52 Oshika, Y. et al. (1998) Thrombospondin 2 gene expression is correlated with decreased vascularity in non-small cell lung cancer. Clin. Cancer Res. 4, 1785–1788 53 Hsu, S.C. et al. (1996) Inhibition of angiogenesis in human glioblastomas by chromosome 10 induction of thrombospondin-1. Cancer Res. 56, 5684–5691 54 Kazuno, M. et al. (1999) Thrombospondin-2 (TSP2) expression is inversely correlated with vascularity in glioma. Eur. J. Cancer 35, 502–506 55 Tokunaga, T. et al. (1999) Thrombospondin 2 expression is correlated with inhibition of angiogenesis and metastasis of colon cancer. Br. J. Cancer 79, 354–359 56 Souchelnitskiy, S. et al. (1995) Thrombospondins selectively activate one of the two latent forms of
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
407
transforming growth factor-β present in adrenocortical cell-conditioned medium. Endocrinology 136, 5118–5126 Yehualaeshet, T. et al. (1999) Activation of rat alveolar macrophage-derived latent transforming growth factor β-1 by plasmin requires interaction with thrombospondin-1 and its cell surface receptor, CD36. Am. J. Pathol. 155, 841–851 Crawford, S.E. et al. (1998) Thrombospondin-1 is a major activator of TGF-β1 in vivo. Cell 93, 1159–1170 Abdelouahed, M. et al. (2000) Activation of platelet transforming growth factor-β 1 in the absence of thrombospondin-1. J. Biol. Chem. 275, 17933–17936 Folkman, J. and D’Amore, P.A. (1996) Blood vessel formation: what is its molecular basis? Cell 87, 1153–1155 Andreasen, P.A. et al. (2000) The plasminogen activation system in tumor growth, invasion, and metastasis. Cell. Mol. Life Sci. 57, 25–40 Tuszynski, G.P. et al. (1992) Thrombospondin levels in patients with malignancy. Thromb. Haemost. 67, 607–611 Nathan, F.E. et al. (1994) Plasma thrombospondin levels in patients with gynecologic malignancies. Cancer 73, 2853–2858 Yamashita, Y. et al. (1998) Plasma thrombospondin levels in patients with colorectal carcinoma. Cancer 82, 632–638 Molema, G. et al. (1998) Tumor vasculature targeted therapies: getting the players organized. Biochem. Pharmacol. 55, 1939–1945 Weinstat-Saslow, D.L. et al. (1994) Transfection of thrombospondin 1 complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis. Cancer Res. 54, 6504–6511 Volpert, O.V. et al. (1998) A human fibrosarcoma inhibits systemic angiogenesis and the growth of experimental metastases via thrombospondin-1. Proc. Natl. Acad. Sci. U. S. A. 95, 6343–6348 Bleuel, K. et al. (1999) Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization. Proc. Natl. Acad. Sci. U. S. A. 96, 2065–2070 Streit, M. et al. (1999) Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas. Am. J. Pathol. 155, 441–452 Streit, M. et al. (1999) Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis. Proc. Natl. Acad. Sci. U. S. A. 96, 14888–14893 Bogdanov, A., Jr et al. (1999) Treatment of experimental brain tumors with thrombospondin-1 derived peptides: an in vivo imaging study. Neoplasia 1, 438–445 Castle, V. et al. (1991) Antisense-mediated reduction in thrombospondin reverses the malignant phenotype of a human squamous carcinoma. J. Clin. Invest. 87, 1883–1888 Wang, T.N. et al. (1996) Inhibition of breast cancer progression by an antibody to a thrombospondin-1 receptor. Surgery 120, 449–454 Maeda, K. et al. (2000) Expression of vascular endothelial growth factor and thrombospondin-1 in colorectal carcinoma. Int. J. Mol. Med. 5, 373–378 Nicosia, R.F. and Tuszynski, G.P. (1994) Matrix-bound thrombospondin promotes angiogenesis in vitro. J. Cell Biol. 124, 183–193