R Benezra P de Candia H Li E Romero D Lyden S Rafii and M Ruzinova

*Department of Cell Biology and fDepartment of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021; fDepartment of Medicine-Hematology/Oncology, New York Medical Center, New York, New York 10021

There is a widely held belief in the field of cancer biology, proposed originally by Dr. Judah Folkman, that tumors cannot grow beyond a very limited size if the mi-crovessel density within the tumor is sufficiently low. Therefore, inhibition of the formation of blood vessels into the tumor bed is likely to be an effective anticancer therapy. With a large number of antiangiogenic drugs already in the clinic, the results beginning to trickle in suggest that this first generation of therapeutics might not be all that was hoped for. Although it is too early to draw firm conclusions, we can at least begin to think of ways to improve the effectiveness of these therapies.

We have taken a genetic approach to the dissection of the effects of antiangiogenesis on tumor growth in mouse models. We have done this by the disruption of two genes (Id1 and Id3) shown to be essential for tumor angiogenesis but dispensable for normal angiogenic processes (Ly-den et al. 1999). We have characterized the effects of loss of these gene products on the growth and metastasis of tumors initiated by subcutaneous inoculation, by overexpression of oncogenes, and by the loss of tumor suppressor genes. These systems have demonstrated quite clearly that there is a broad distribution of effects on the growth of a tumor that results from antiangiogenic interventions. Some tumors respond as predicted by showing lower growth after hypoxic stress; others, however, grow to rather large sizes despite extensive hemorrhage and necrosis internal to the tumor mass by what appears to be an effective utilization of peripheral vasculature. A viable rim of cells that surround a necrotic core is an attractive target for chemotherapeutic intervention and points to the likelihood that antiangiogenic and anti-cell-based therapies will synergize to inhibit the growth of tumors. Finally, the Id knockout model, by providing an animal in which tumor vasculature is normally blocked, has allowed unequivocal demonstration that bone-marrow-derived precursor cells are critical components in the establishment of a functional tumor vasculature (Lyden et al. 2001). These results, overall, have significant implications both in the basic biology of tumor vascularization and in the application to antiangiogenic intervention.

BASIC BIOLOGY OF THE Id PROTEINS

How was it determined that the Id proteins are critical components of tumor angiogenesis? The history of this discovery provides an excellent example of how the path from basic science to clinical implication can be unexpectedly direct. The Id proteins were originally defined as naturally occurring dominant negative antagonists of the basic-helix-loop-helix (bHLH) family of transcription factors (for review, see Benezra 2001). The bHLH proteins contain a cluster of amino acids rich in basic residues adjacent to an HLH dimerization motif that mediates DNA binding of homodimeric and heterodimeric bHLH complexes. Because Id lacks the basic DNA-bind-ing domain, heterodimers between Id and bHLH proteins cannot bind DNA (see Fig. 1). This dominant negative mode of inhibition of DNA-binding activity is widely used in the cell, as it is also employed by members of the leucine zipper and homeodomain protein families (Treacy et al. 1991; Ron and Habener 1992).

Biochemical and genetic data have established that the primary method whereby Id exerts its dominant negative effect is to sequester the ubiquitously expressed bHLH proteins referred to as E proteins and to prevent them from binding DNA either alone or as heterodimers with tissue-restricted bHLH proteins. Specifically, endogenous Id1 protein present in an undifferentiated myoblast quantitatively sequesters the available pool of E2A proteins, forces dimers between MyoD and E47 (a splice product of the E2A gene) to resist Id1 inhibition, and causes the transcriptional inhibitory effects of Id1 and Id3 to be overcome by the overexpression of E proteins. In addition, the postnatal lethality observed in mice lacking products of the E2A gene is partially suppressed by disruption of the Id1 gene. Thus, by sequestering E proteins that are required to enhance transcriptional activity of bHLH proteins in multiple cell types, the Id proteins can control the activity of bHLH proteins in diverse lineages.

Id1 and Id3 are co-expressed temporally and spatially during murine neurogenesis and angiogenesis, two processes that are affected in the Id1/Id3 double knockout animals (Jen et al. 1997; Lyden et al. 1999). In general terms, Id1, 2, and 3 are expressed in dividing neuroblasts in the central nervous system (CNS) up to about E12.5, after which time Id2 expression appears in the presumptive neurons that were undergoing maturation in both the future cerebellum and the cerebral cortex. Id4, which, unlike the other Id genes is localized exclusively in the CNS and peripheral nervous system, has a pattern of expression that is often complementary to Id1 and Id3 and is

Figure 1. Mechanism of Id activity. The primary target of the Id proteins in the cell are the E proteins (E) which homodimerize in lymphoid cells via their helix-loop-helix (HLH) domains and bind negatively charged DNA through contacts with the two basic clusters of amino acids that carry positive charges under physiological conditions. E proteins also heterodimerize with other bHLH proteins in many nonlymphoid tissues and bind DNA similarly. Id can associate with E proteins via its HLH domain, but because it lacks a basic region, the E-Id heterodimer is incapable of binding DNA. Geometries depicted are used to show the essential features of the model. (Reprinted, with permission, from Benezra 2001 [copyright Elsevier Science] Online permission pending.)

Figure 1. Mechanism of Id activity. The primary target of the Id proteins in the cell are the E proteins (E) which homodimerize in lymphoid cells via their helix-loop-helix (HLH) domains and bind negatively charged DNA through contacts with the two basic clusters of amino acids that carry positive charges under physiological conditions. E proteins also heterodimerize with other bHLH proteins in many nonlymphoid tissues and bind DNA similarly. Id can associate with E proteins via its HLH domain, but because it lacks a basic region, the E-Id heterodimer is incapable of binding DNA. Geometries depicted are used to show the essential features of the model. (Reprinted, with permission, from Benezra 2001 [copyright Elsevier Science] Online permission pending.)

found in regions undergoing neuronal maturation. In adult animals, Id1 and Id3 are no longer expressed in the brain, but Id2 expression persists in the Purkinje cells of the cerebellum and to a lesser extent in the mitral cells of the olfactory bulb in layer five of the neocortex (Neuman et al. 1993; Jen et al. 1997). Given the similarities and biochemical activity of Id1, 2, and 3 and the known role of bHLH proteins in neural development, it was not surprising to observe premature neural differentiation in Id1,3 knockout animals when Id2 expression was lost in dividing neuroblasts (~E12.5) (Lyden et al. 1999).

A role for Id in angiogenesis was unexpected, however. Angiogenesis, the branching and spreading of capillaries from the primary vascular plexus, occurs both in the yolk sac and in the embryo proper, particularly in the brain. Signaling from both the VEGF and Tie-2 receptors has been implicated in the process as well as during tumor an-giogenesis (for review, see Yancopoulos et al. 2000). Little is known of the involvement of bHLH proteins, however, during these processes. bHLH proteins that contain a second dimerization motif (referred to as a PAS domain) are expressed in endothelial cells (HIF-1a, EPAS, HRF) but are unlikely to be regulated by Id, as the specificity of these proteins is dictated by the PAS motif and no HLH/bHLH-PAS interactions have been documented. More importantly, HIF-1 a and EPAS have been shown to up-regulate VEGF expression (for review, see Levy et al. 1997), and loss of Id function leads to a decrease in VEGF expression, implying, counter to its normal mode of action, that Id would enhance the activity of these tran scription factors directly or indirectly. bHLH-EC2 is expressed in endothelial cells and lacks a PAS domain (Quertermous et al. 1994), making it a possible candidate for Id regulation. At least one copy of the Id1 or Id3 genes is required in mice to prevent embryonic lethality associated with premature neural differentiation and angiogenic defects in the brain. Interestingly, Id1 and Id3 but not Id2 are expressed in the endothelial cells in the brain whereas Id1, 2, and 3 are all expressed in endothelial cells throughout the rest of the embryo during development, perhaps accounting for the brain specificity of the pheno-type (Lyden et al. 1999). Lethality may be due to intraventricular hemorrhage, which contributes to death in humans and rodents.

ROLE OF Id IN ANGIOGENESIS WITHIN TUMORS

Id1 and 3 expression has been detected in the endothe-lial cells of vessels invading tumors but not in resting vessels within normal tissues (Lyden et al. 1999). This observation, coupled with the genetics described above, led us to examine the Id knockout mice for their ability to support the growth of tumor xenografts. Strikingly, three different tumors fail to grow and/or metastasize in Id1+/-Id3-/- animals (see Fig. 2), and any tumor growth present showed poor vascularization and extensive necrosis. Blood vessels in Id knockout animals lack the ability to branch and sprout and form a normal-caliber lumen during tumor progression. These processes depend on the

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