- Email: [email protected]
P53 and angiogenesis
BB
Biochi~ic~a
|
et Biophysica A~ta
ELSEVIER
Biochimica et Biophysica Acta 1287 (1996) 63-66
Opinion Piece
P53 and angiogenesis Noel Bouck
*
Department of Microbiology-Immunology and R.H. Lurie Cancer Center, Northwestern Univer~'iO'Medical School, 303 East Chicago Avenue, Chicago, 1L 60611, USA Received 18 January 1996; accepted 12 February 1996
The p53 gene (see [1-5]) occupies a singular position among the tumor suppressor genes. It is inactivated with startlingly high frequency in most types of human cancers, and its sole essential function in normal cells seems to be to prevent them from developing into tumors. Although occasional spontaneous [6] and induced [7] embryological defects have been seen in p53 null mice, the majority of such animals develop normally throughout a life span that is limited only by the early onset of malignancies [8]. The protein encoded by the wild type p53 gene prevents cells from progressing towards malignancy in three general ways: (i) by retarding the accumulation of the DNA lesions that underlie tumor progression. In many cell types the presence of wild type p53 guards against genetic damage by insuring that cells with damaged DNA either halt their progression through the cell cycle [1] and repair damage efficiently [9] or are eliminated by apoptosis [5]. (ii) by restricting the exponential growth of incipient tumor cells. Overexpression of wild type p53 can halt the growth of cell lines derived from numerous different human tumors that lack wild type p53 (ex. [10]) and can impose linear stem cell kinetics in a mouse tumor line [11]. Ambient levels of p53 protein from endogenous genes can enforce senescence in oncogene-transformed hematopoietic cells [12], enhance hypoxia driven apoptosis [13] or assist in TGF-B-induced growth arrest [14]. (iii) by preventing the developing tumor from attracting new blood uessels essential for its progressive growth and efficient metastasis in vivo [15]. This short review will focus on the ability of wild type p53 to restrict the ability of cells that express it to induce angiogenesis. The development of an angiogenic phenotype is an essential component of tumor progression. Small tumors that are unable to invoke the vigorous ingrowth of nourish-
* Corresponding [email protected].
author.
Fax:
+1
(312)
9081372;
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ing new vessels remain dormant at the clinically benign size of a few mm [15,16]. Large progressively growing tumors attract the vessels they need by secreting one or more potent angiogenic factors such as bFGF, I1-8, VEGF and TGF-B. Such tumors arise from normal cells, many of which are actually antiangiogenic and actively secrete high levels of molecules that inhibit neovascularization. As the induction of new vessels depends on the relative amounts of inducers and inhibitors in the environment around the vascular endothelial cells [15], the development of inhibitory normal cells into angiogenic tumor cells seems to entail both the down regulation of the secretion of molecules that inhibit angiogenesis and the upregulation of secretion of active inducers of neovascularization. In vitro, both fibroblasts and keratinocytes progressing to tumorigenicity develop an angiogenic phenotype in a step-wise process, during which the secretion of inducers rises and the production of inhibitors falls ([15,17,18]; Volpert and Bouck, unpublished data; Lingen and Bouck, unpublished data). A similar increase in angiogenic activity during tumor progression can also be seen in vivo [19]. Occasionally, tumors continue to produce inhibitors. The activity of such inhibitors is presumably masked in the immediate environment around a tumor by high levels of inducers, but if tumor-derived inhibitors are more stable in the circulation than inducers, they may create an antiangiogenic environment at distant sites, holding in check small metastases from the original tumor [20,21]. Considerable evidence now indicates that, in addition to its well documented contributions to genetic instability and permissive growth, loss of the wild type p53 tumor suppressor gene can contribute to the development of an angiogenic phenotype. Loss of wild type p53 may stimulate the production of a powerful inducer of angiogenesis, VEGF. Overproduction of wild type p53 decreased endogenous VEGF mRNA in 293 cells by about 5-fold and suppressed an ectopic VEGF promoter in several other tumor cells [22], suggesting that loss of wild type p53 function may result in increased secretion of this potent
64
N. Bouck / Biochimica et Biophysica Acre 1287 (l 996) 63-66
angiogenic factor. This expectation is consistent with the enhanced sensitivity of certain tumor cells to VEGF induction by TPA which is noted upon overproduction of a mutant p53 species capable of dominant negative behavior in NIH/3T3 cells [23]. Relief of VEGF suppression by wild type p53 could also be responsible for the significant increase in total angiogenesis inducing activity (which can be neutralized by anti-VEGF antibodies, Volpert and Bouck, unpublished data) observed when fibroblasts cultured from Li Fraumeni patients become immortal and, in the process, lose their single wild type p53 allele [18]. P53 is only one of several influences on VEGF secretion. Activated ras is a potent inducer of VEGF in epithelial cells [24,25] and in human fibroblasts (Volpert, Dameron and Bouck, unpublished data). When immortal Li Fraumeni fibroblasts that have already lost wild type p53 are induced to progress in vitro to tumorigenicity by transfection with activated ras, VEGF secretion again increases, and its effect is additive with the increase that occurs upon immortalization and loss of wild type p53. Wild type p53 can also limit the angiogenic phenotype of cells by modulating the production of angiogenesis inhibitors. Whereas wild type p53 inhibits the production of angiogenic VEGF, as might be expected of a tumor suppressor, it also stimulates the production of inhibitors of angiogenesis. Restoring wild type p53 to tumor cells that have lost it can affect two distinct inhibitors produced by two different cell types. When restored to a human glioblastoma line, wild type p53 function stimulated the secretion of an unidentified protein inhibitor of angiogenesis and, as a result, the cells evolved from being potently angiogenic to antiangiogenic [26]. When restored to a human breast carcinoma line, wild type p53 also caused these cells to switch from angiogenic to antiangiogenic, in this case due to the increased secretion of a known inhibitor of angiogenesis, thrombospondin [27]. In each case, the p53-producing cells synthesized significant quantities of an inhibitor(s). Indeed, when their secreted proteins were mixed with equal amounts of protein secreted by the relevant angiogenic parental tumor line, the mixture inhibited the migration of capillary endothelial cells in vitro and blocked neovascularization towards the secreted proteins from the angiogenic parent in vivo. Wild type p53 is only one of several human tumor suppressor genes that can cause tumor cells to switch from an angiogenic to an antiangiogenic phenotype due to the production of an inhibitor (see [15]). Others include the retinoblastoma gene and unidentified tumor suppressor genes on chromosomes 10 and 17. A third cell type in which p53 influences angiogenesis by stimulating an inhibitor is the fibroblast. Wild type p53 supports the secretion of inhibitory thrombospondin-1 by fibroblasts cultured either from normal individuals or from Li Fraumeni patients who have only a single wild type p53 allele [17,18]. When the wild type p53 allele was lost upon
immortalization of the Li Fraumeni fibroblasts, thrombospondin-1 secretion fell dramatically, in concert with an increase in VEGF production. The cells, in turn, switched from antiangiogenic to angiogenic. Analysis of the effects of specific neutralizing antibodies have shown that the fall in thrombospondin was necessary and sufficient for this switch. This thrombospondin-dependent, antiangiogenic phenotype of mortal fibroblasts is a product of wild type p53 function, for (i) it can be re-established in immortal fibroblasts in the absence of any major change in total inducing activity, simply by re-introducing a wild type p53 allele [17,18] and (ii) it is present in wild type mouse embryo fibroblasts but absent from fibroblasts cultured, in parallel, from p53 null embryos (Volpert, Dameron and Bouck, unpublished data). In fibroblasts, wild type p53 seems to stimulate the thrombospondin gene at the level of transcription, in part via a p53 response element in its first intron. When this element was mutated, an ectopic thrombospondin-I promoter lost a significant fraction of its p53 sensitivity (Stellmach and Bouck, unpublished data). Experiments describing p53 support of the secretion of inhibitors of angiogenesis have all been performed with cultured cells, but they appear to be relevant to natural tumors evolving in vivo. Inhibitors are secreted by cells producing wild type p53 at levels several times greater than those minimally needed to inhibit angiogenesis induced by cells cultured from malignant human tumors ([ 17,18]; Volpert, Dameron and Bouck, unpublished data). During the development of dermal fibrosarcomas in bovine papilloma virus transgenic mice, wild type p53 was lost at the time the tumors first showed signs of vascularization [28]. And p53 is often inactivated, either directly or as a result of MDM-2 amplification, in primary human soft tissue sarcomas [29] where its loss of function is linked to a high proliferation index and a poor clinical outcome [30]. In one set of operable, invasive breast cancers, p53 expression (usually indicative of the presence of a mutation) and tumor angiogenesis were, each, powerful predictors of recurrence [31]. A direct test of the in vivo relevance of p53 control of angioinhibitory thrombospondin is now possible with the development of thrombospondin-1 knock-out mice (J. Lawler, personal communication). If loss of thrombospondin-I mediated by loss of wild type p53 is rate limiting in the formation of some tumor types, one would expect that, in p53 + mice, such tumors would arise sooner, with increased frequency, and, possibly, without loss of the remaining wild type p53 allele, in the animals unable to produce thrombospondin-I compared to those which can produce it. Such a test is under way. Gain in angiogenic activity upon loss of wild type p53 can provide cells with a selective advantage, as, for example, when a clone of cells in one portion of a carcinoma in situ loses wild type p53 and with it the ability to secrete high levels of inhibitors. Since the bulk flow of secreted
N. Bouck / Biochimica et Biophysica Acre 1287 (1996) 63-66
proteins moves outward from tumors [32], lack of a tumor-derived angiogenesis inhibitor in one sector of a tumor mass should result in increased neovascularization to this region and subsequent overgrowth of the better vascularized cells. In Li Fraumeni patients, malignancies of the breast, brain and connective tissues predominate, although all tissues carry the mutant p53 allele [4,33]. The reason for the tissue specificity is not clear. The ability of wild type p53 to influence the angiogenic phenotype of some, but not all cells might contribute to this mysterious aspect of p53 biology. All of the tissues and cell types for which wild type p53 has been shown to affect neovascularization (breast, brain and connective tissue, but not keratinocytes) happen to be prone to tumors in Li Fraumeni patients. Could it be that only in Li-Fraumeni tumor-prone tissues does loss of wild type p53 significantly enhance angiogenesis? Although primary data are incomplete, the observations concerning antiangiogenic activities of wild type p53 are subject to the same constraints and caveats that limit analysis of other anti-tumor activities of p53. Control of angiogenesis by p53 is context dependent within a single lineage, as are other functions such as apoptosis and growth control [5,34,35]. The introduction of wild type p53 successfully abrogated the angiogenic phenotype in one human breast cell line, but not in another (Volpert, Stellroach and Bouck, unpublished data). P53 control of angiogenesis is also cell type-specific. The wild type protein effectively supported the synthesis of thrombospondin in fibroblasts, but not in glioblastoma cells where its overexpression had no effect on thrombospondin, although it did induce a different inhibitor of angiogenesis ([26]; Cavenee and Bouck, unpublished data). While loss of wild type p53 means loss of angiogenesis inhibitory activity in fibroblasts, it had no effect on inhibitory activity secreted by human keratinocytes (Lingen and Bouck, unpublished data). Control of angiogenesis by p53 can be overridden. Just as enhanced myb gene expression overrode growth arrest by p53 [36], p53-mediated antiangiogenesis can also be overcome. For example, the HT1080 human fibrosarcoma line retains wild type p53 and produces high levels of active, antiangiogenic thrombospondin. However, the cells are still angiogenic and tumorigenic, because the inhibitory effects of thrombospondin are overcome by the secretion of very high levels of several inducers of angiogenesis (Volpert and Bouck, unpublished data). The antiangiogenic activity of wild type p53 is especially interesting and relevant to the clinical situation, because it has the potential to create a field effect capable of influencing nearby tumor cells that may not produce wild type p53. Other tumor suppressor actions of p53, although influenced by the cellular environment (for example [14,37]), are cell autonomous. In contrast, the antiangiogenic activity of wild type p53 depends on a secreted
65
protein acting at a distance and thus might provide a useful bystander effect in therapeutic protocols designed to reestablish wild type p53 in tumors. The fact that fibroblasts containing a single wild type p53 allele secrete four times the thrombospondin needed to inhibit angiogenesis induced by an equivalent number of tumor cells (Volpert and Bouck, unpublished data), leaves open the possibility that re-establishment of wild type p53 in a minority of such tumor cells in vivo could, by limiting neovascularization. curtail the growth of the whole tumor. There are clear challenges ahead for research on the antiangiogenic aspects of p53 function. First, it will be important to establish that p53 control of angiogenesis does indeed play a role in the genesis of spontaneous human cancers. Secondly, if this mechanism is vital to the development of some human tumors, it will be important to identify the relevant p53-dependent angiogenesis inhibitors and to determine whether they are useful clinically in neutralizing the enhanced neovascularization associated with loss of p53 function.
Acknowledgements I am grateful to the NIH for funding that supports work in my laboratory on p53 (CA642309) and to Roni Stellmach and Olga Volpert for critical reading of this manuscript.
References [1] Donehower, L.A, and Bradley. A. (1993) Biochim. Biophys. Acta 1155, 181-205. [2] Levine, A.J. (1993) Annu. Rev. Biochem. 62, 623-651. [3] Lane, D.P. (1994) Brit. Med. Bull. 50, 582-599. [4] Malkin, D. (1994) Biochim. Biophys. Acta 1198, 197-213. [5] Haffner, R. and Oren, M. (1995) Curr. Opinion Genet. Dev. 5. 84-94. [6] Sah.V.P., Attardi, L.D., Mulligan, G.J., Williams, B.O., Bronson, R.T. and Jacks, T. (1995) Nature Genet. 10, 175-179. [7] Nicol, C.J, Harrison, M.L., Laposa. R.R., Gimelshtein, I.L. and Wells, P.G. (1995) Nature Genet. 10. 181-187. [8] Donehower, L.A., Harvey, M., Slagle, B.L.. McArthur, M.J., Montgomery, C,A., Butel, J.S. and Bradley, A. (1992) Nature 356, 215-221. [9] Marks, J. (1994) Science 266, 1321-1322. [10] Ultrich, S.J, Anderson, C.W., Mercer, W.E, and Appella, E. (1992) J. Biol. Chem. 267, 15259-15262. [11] Sherley, J.L., Stadler, P.B. and Johnson. D.R. (1995) Proc. Natl. Acad. Sci. USA 92, 136-140. [12] Metz, T., Harris, A.W. and Adams, J.M. (1995) Cell 82, 29-36. [131 Graeber, T.G., Osmanian, C., Jacks, T., Housmam D.E., Koch, C.J.. Lowe, S.W. and Giaccia. A.J. (1996) Nature 379, 88-91. [14] Ewen, M.E., Oliver, CJ,, Sluss, H.K., Miller, S.J. and Peeper, D.S. (1995) Genes Dev. 9, 204-217. [15] Bouck. N., Stellmach, V. and Hsu, S. (1996) Adv. Cancer Res., in press, [16] Folkmam J. (1995) in The Molecular Basis of Cancer, Mendelshon, J., HoMey, P.M., Israel, M.A. and Liotta. L.A., eds. W.B. Saunders, pp. 206-232.
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[17] Dameron, K.M., Volpert, O.V., Tainsky, M.A. and Bouck, N. (1994) Science 265, 1582-1584. [18] Dameron, K.M., Volpert, O.V., Tainsky, M.A. and Bouck, N. (1994) Cold Spring Harbor Symp. Quant. Biol. LIX, 483-489. [19] Bossi, P., Viale, G., Lee, A.K.C., Alfano, R.M., Coggi, G. and Bosari, S. (1995) Cancer Res. 55, 5049-5053. [20] O'Reilly, M.S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R.A., Moses, M., Lane, W.S., Cao, Y., Sage, E.H. and Folkman, J. (1994) Cell 79, 315-328. [21] Holmgren, L., O'Reilly, M.S. and Folkman, J. (1995) Nature Med. 1, 149-153. [22] Mukhopadhyay, D., Tsiokas, L. and Sukhatme, V.P. (1995) Cancer Res. 55, 6161-6165. [23] Kieser, A., Weich, H.A., Brandner, G., Marme, D. and Kolch, W. (1994) Oncogene 9, 963-969, [24] Rak, J., Mitsuhashi, Y., Bayko, L., Filmus, J., Shirasawa, S., Sasazuki, T. and Kerbel, R.S. (1995) Cancer Res. 55, 4575-4580. [25] Grugel, S., Finkenzeller, G., Weindel, K., Barelon, B. and Marme, D. (1995) J. Biol. Chem. 270, 25915-25919. [26] Van Meir, E.G., Polverini. P.J., Chazin, V.R., Huang, H.-J.S., de Tribolet, N. and Cavenee, W.K. (1994) Nature Genet. 8, 171-176. [27] Volpert, O.V., Stellmach, V. and Bouck, N. (1995) Breast Cancer Res. Treat. 36, 119-126.
[28] Christofori, G. and Hanahan, D. (1994) Cancer Biol. 5, 3-12. [29] Leach, F.S., Tokino, T., Meltzer, P., Burrell, M., Oliner, J.D., Smith, S., Hill, D.E., Sidransky, D., Kinzler, K.W. and Vogelstein, B. (1993) Cancer Res. 53,2231-2234. [30] Drobnjak, M., Latres, E., Pollack, D., Karpeh, M., Dudas, M., Woodruff, J.M., Brennan, M.F. and Cordon-Cardo, C. (1994) J. Natl. Cancer Inst. 86, 549-554. [31] Gasparini, G., Weidner, N., Bevilacqua, P., Maluta, S., Palma, P.D., Caffo, O., Barbareschi, M., Boracchi, P., Marubini, E. and Pozza, F. (1994) J. Clin. Oncol. 12, 454-466. [32] DiResta, G.R., Lee, J., Larson, S.M. and Arbit, E. (1993) Microvasc. Res. 46, 158-177. [33] Li, F.P, and Fraumeni, J.F. (1982) JAMA 247, 2692-2694. [34] Lin, Y. and Benchimol, S. (1995) Mol. Cell. Biol. 15, 6045-6054. [35] Deppert, W. (1994) Cancer Biol. 5,187-202. [36] Lin, D., Fiscella, M., O"Connor, P.M., Jackman, J., Chen, M., Lno, L.L., Sala, A., Travali, S., Appella, E. and Mercer, W,E. (1994) Proc, Natl. Acad. Sci. USA 91, 10079-10083. [37] Canman, C.E., Gilmer, T.M., Coutts, S.B. and Kastan, M.B, (1995) Genes Dev. 9, 600-611.
Biochi~ic~a
|
et Biophysica A~ta
ELSEVIER
Biochimica et Biophysica Acta 1287 (1996) 63-66
Opinion Piece
P53 and angiogenesis Noel Bouck
*
Department of Microbiology-Immunology and R.H. Lurie Cancer Center, Northwestern Univer~'iO'Medical School, 303 East Chicago Avenue, Chicago, 1L 60611, USA Received 18 January 1996; accepted 12 February 1996
The p53 gene (see [1-5]) occupies a singular position among the tumor suppressor genes. It is inactivated with startlingly high frequency in most types of human cancers, and its sole essential function in normal cells seems to be to prevent them from developing into tumors. Although occasional spontaneous [6] and induced [7] embryological defects have been seen in p53 null mice, the majority of such animals develop normally throughout a life span that is limited only by the early onset of malignancies [8]. The protein encoded by the wild type p53 gene prevents cells from progressing towards malignancy in three general ways: (i) by retarding the accumulation of the DNA lesions that underlie tumor progression. In many cell types the presence of wild type p53 guards against genetic damage by insuring that cells with damaged DNA either halt their progression through the cell cycle [1] and repair damage efficiently [9] or are eliminated by apoptosis [5]. (ii) by restricting the exponential growth of incipient tumor cells. Overexpression of wild type p53 can halt the growth of cell lines derived from numerous different human tumors that lack wild type p53 (ex. [10]) and can impose linear stem cell kinetics in a mouse tumor line [11]. Ambient levels of p53 protein from endogenous genes can enforce senescence in oncogene-transformed hematopoietic cells [12], enhance hypoxia driven apoptosis [13] or assist in TGF-B-induced growth arrest [14]. (iii) by preventing the developing tumor from attracting new blood uessels essential for its progressive growth and efficient metastasis in vivo [15]. This short review will focus on the ability of wild type p53 to restrict the ability of cells that express it to induce angiogenesis. The development of an angiogenic phenotype is an essential component of tumor progression. Small tumors that are unable to invoke the vigorous ingrowth of nourish-
* Corresponding [email protected].
author.
Fax:
+1
(312)
9081372;
e-mail:
n-
0304-419X/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S 0 3 0 4 - 4 1 9 X ( 9 6 ) 0 0 0 0 5 - 4
ing new vessels remain dormant at the clinically benign size of a few mm [15,16]. Large progressively growing tumors attract the vessels they need by secreting one or more potent angiogenic factors such as bFGF, I1-8, VEGF and TGF-B. Such tumors arise from normal cells, many of which are actually antiangiogenic and actively secrete high levels of molecules that inhibit neovascularization. As the induction of new vessels depends on the relative amounts of inducers and inhibitors in the environment around the vascular endothelial cells [15], the development of inhibitory normal cells into angiogenic tumor cells seems to entail both the down regulation of the secretion of molecules that inhibit angiogenesis and the upregulation of secretion of active inducers of neovascularization. In vitro, both fibroblasts and keratinocytes progressing to tumorigenicity develop an angiogenic phenotype in a step-wise process, during which the secretion of inducers rises and the production of inhibitors falls ([15,17,18]; Volpert and Bouck, unpublished data; Lingen and Bouck, unpublished data). A similar increase in angiogenic activity during tumor progression can also be seen in vivo [19]. Occasionally, tumors continue to produce inhibitors. The activity of such inhibitors is presumably masked in the immediate environment around a tumor by high levels of inducers, but if tumor-derived inhibitors are more stable in the circulation than inducers, they may create an antiangiogenic environment at distant sites, holding in check small metastases from the original tumor [20,21]. Considerable evidence now indicates that, in addition to its well documented contributions to genetic instability and permissive growth, loss of the wild type p53 tumor suppressor gene can contribute to the development of an angiogenic phenotype. Loss of wild type p53 may stimulate the production of a powerful inducer of angiogenesis, VEGF. Overproduction of wild type p53 decreased endogenous VEGF mRNA in 293 cells by about 5-fold and suppressed an ectopic VEGF promoter in several other tumor cells [22], suggesting that loss of wild type p53 function may result in increased secretion of this potent
64
N. Bouck / Biochimica et Biophysica Acre 1287 (l 996) 63-66
angiogenic factor. This expectation is consistent with the enhanced sensitivity of certain tumor cells to VEGF induction by TPA which is noted upon overproduction of a mutant p53 species capable of dominant negative behavior in NIH/3T3 cells [23]. Relief of VEGF suppression by wild type p53 could also be responsible for the significant increase in total angiogenesis inducing activity (which can be neutralized by anti-VEGF antibodies, Volpert and Bouck, unpublished data) observed when fibroblasts cultured from Li Fraumeni patients become immortal and, in the process, lose their single wild type p53 allele [18]. P53 is only one of several influences on VEGF secretion. Activated ras is a potent inducer of VEGF in epithelial cells [24,25] and in human fibroblasts (Volpert, Dameron and Bouck, unpublished data). When immortal Li Fraumeni fibroblasts that have already lost wild type p53 are induced to progress in vitro to tumorigenicity by transfection with activated ras, VEGF secretion again increases, and its effect is additive with the increase that occurs upon immortalization and loss of wild type p53. Wild type p53 can also limit the angiogenic phenotype of cells by modulating the production of angiogenesis inhibitors. Whereas wild type p53 inhibits the production of angiogenic VEGF, as might be expected of a tumor suppressor, it also stimulates the production of inhibitors of angiogenesis. Restoring wild type p53 to tumor cells that have lost it can affect two distinct inhibitors produced by two different cell types. When restored to a human glioblastoma line, wild type p53 function stimulated the secretion of an unidentified protein inhibitor of angiogenesis and, as a result, the cells evolved from being potently angiogenic to antiangiogenic [26]. When restored to a human breast carcinoma line, wild type p53 also caused these cells to switch from angiogenic to antiangiogenic, in this case due to the increased secretion of a known inhibitor of angiogenesis, thrombospondin [27]. In each case, the p53-producing cells synthesized significant quantities of an inhibitor(s). Indeed, when their secreted proteins were mixed with equal amounts of protein secreted by the relevant angiogenic parental tumor line, the mixture inhibited the migration of capillary endothelial cells in vitro and blocked neovascularization towards the secreted proteins from the angiogenic parent in vivo. Wild type p53 is only one of several human tumor suppressor genes that can cause tumor cells to switch from an angiogenic to an antiangiogenic phenotype due to the production of an inhibitor (see [15]). Others include the retinoblastoma gene and unidentified tumor suppressor genes on chromosomes 10 and 17. A third cell type in which p53 influences angiogenesis by stimulating an inhibitor is the fibroblast. Wild type p53 supports the secretion of inhibitory thrombospondin-1 by fibroblasts cultured either from normal individuals or from Li Fraumeni patients who have only a single wild type p53 allele [17,18]. When the wild type p53 allele was lost upon
immortalization of the Li Fraumeni fibroblasts, thrombospondin-1 secretion fell dramatically, in concert with an increase in VEGF production. The cells, in turn, switched from antiangiogenic to angiogenic. Analysis of the effects of specific neutralizing antibodies have shown that the fall in thrombospondin was necessary and sufficient for this switch. This thrombospondin-dependent, antiangiogenic phenotype of mortal fibroblasts is a product of wild type p53 function, for (i) it can be re-established in immortal fibroblasts in the absence of any major change in total inducing activity, simply by re-introducing a wild type p53 allele [17,18] and (ii) it is present in wild type mouse embryo fibroblasts but absent from fibroblasts cultured, in parallel, from p53 null embryos (Volpert, Dameron and Bouck, unpublished data). In fibroblasts, wild type p53 seems to stimulate the thrombospondin gene at the level of transcription, in part via a p53 response element in its first intron. When this element was mutated, an ectopic thrombospondin-I promoter lost a significant fraction of its p53 sensitivity (Stellmach and Bouck, unpublished data). Experiments describing p53 support of the secretion of inhibitors of angiogenesis have all been performed with cultured cells, but they appear to be relevant to natural tumors evolving in vivo. Inhibitors are secreted by cells producing wild type p53 at levels several times greater than those minimally needed to inhibit angiogenesis induced by cells cultured from malignant human tumors ([ 17,18]; Volpert, Dameron and Bouck, unpublished data). During the development of dermal fibrosarcomas in bovine papilloma virus transgenic mice, wild type p53 was lost at the time the tumors first showed signs of vascularization [28]. And p53 is often inactivated, either directly or as a result of MDM-2 amplification, in primary human soft tissue sarcomas [29] where its loss of function is linked to a high proliferation index and a poor clinical outcome [30]. In one set of operable, invasive breast cancers, p53 expression (usually indicative of the presence of a mutation) and tumor angiogenesis were, each, powerful predictors of recurrence [31]. A direct test of the in vivo relevance of p53 control of angioinhibitory thrombospondin is now possible with the development of thrombospondin-1 knock-out mice (J. Lawler, personal communication). If loss of thrombospondin-I mediated by loss of wild type p53 is rate limiting in the formation of some tumor types, one would expect that, in p53 + mice, such tumors would arise sooner, with increased frequency, and, possibly, without loss of the remaining wild type p53 allele, in the animals unable to produce thrombospondin-I compared to those which can produce it. Such a test is under way. Gain in angiogenic activity upon loss of wild type p53 can provide cells with a selective advantage, as, for example, when a clone of cells in one portion of a carcinoma in situ loses wild type p53 and with it the ability to secrete high levels of inhibitors. Since the bulk flow of secreted
N. Bouck / Biochimica et Biophysica Acre 1287 (1996) 63-66
proteins moves outward from tumors [32], lack of a tumor-derived angiogenesis inhibitor in one sector of a tumor mass should result in increased neovascularization to this region and subsequent overgrowth of the better vascularized cells. In Li Fraumeni patients, malignancies of the breast, brain and connective tissues predominate, although all tissues carry the mutant p53 allele [4,33]. The reason for the tissue specificity is not clear. The ability of wild type p53 to influence the angiogenic phenotype of some, but not all cells might contribute to this mysterious aspect of p53 biology. All of the tissues and cell types for which wild type p53 has been shown to affect neovascularization (breast, brain and connective tissue, but not keratinocytes) happen to be prone to tumors in Li Fraumeni patients. Could it be that only in Li-Fraumeni tumor-prone tissues does loss of wild type p53 significantly enhance angiogenesis? Although primary data are incomplete, the observations concerning antiangiogenic activities of wild type p53 are subject to the same constraints and caveats that limit analysis of other anti-tumor activities of p53. Control of angiogenesis by p53 is context dependent within a single lineage, as are other functions such as apoptosis and growth control [5,34,35]. The introduction of wild type p53 successfully abrogated the angiogenic phenotype in one human breast cell line, but not in another (Volpert, Stellroach and Bouck, unpublished data). P53 control of angiogenesis is also cell type-specific. The wild type protein effectively supported the synthesis of thrombospondin in fibroblasts, but not in glioblastoma cells where its overexpression had no effect on thrombospondin, although it did induce a different inhibitor of angiogenesis ([26]; Cavenee and Bouck, unpublished data). While loss of wild type p53 means loss of angiogenesis inhibitory activity in fibroblasts, it had no effect on inhibitory activity secreted by human keratinocytes (Lingen and Bouck, unpublished data). Control of angiogenesis by p53 can be overridden. Just as enhanced myb gene expression overrode growth arrest by p53 [36], p53-mediated antiangiogenesis can also be overcome. For example, the HT1080 human fibrosarcoma line retains wild type p53 and produces high levels of active, antiangiogenic thrombospondin. However, the cells are still angiogenic and tumorigenic, because the inhibitory effects of thrombospondin are overcome by the secretion of very high levels of several inducers of angiogenesis (Volpert and Bouck, unpublished data). The antiangiogenic activity of wild type p53 is especially interesting and relevant to the clinical situation, because it has the potential to create a field effect capable of influencing nearby tumor cells that may not produce wild type p53. Other tumor suppressor actions of p53, although influenced by the cellular environment (for example [14,37]), are cell autonomous. In contrast, the antiangiogenic activity of wild type p53 depends on a secreted
65
protein acting at a distance and thus might provide a useful bystander effect in therapeutic protocols designed to reestablish wild type p53 in tumors. The fact that fibroblasts containing a single wild type p53 allele secrete four times the thrombospondin needed to inhibit angiogenesis induced by an equivalent number of tumor cells (Volpert and Bouck, unpublished data), leaves open the possibility that re-establishment of wild type p53 in a minority of such tumor cells in vivo could, by limiting neovascularization. curtail the growth of the whole tumor. There are clear challenges ahead for research on the antiangiogenic aspects of p53 function. First, it will be important to establish that p53 control of angiogenesis does indeed play a role in the genesis of spontaneous human cancers. Secondly, if this mechanism is vital to the development of some human tumors, it will be important to identify the relevant p53-dependent angiogenesis inhibitors and to determine whether they are useful clinically in neutralizing the enhanced neovascularization associated with loss of p53 function.
Acknowledgements I am grateful to the NIH for funding that supports work in my laboratory on p53 (CA642309) and to Roni Stellmach and Olga Volpert for critical reading of this manuscript.
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