Id Proteins at the Cross-Road of Development and Cancer

Id proteins at the cross-road of development and cancer

Anna Lasorella1, Takuma Uo1 and Antonio Iavarone*,1

1Department of Neurology, Developmental and Molecular Biology and Comprehensive Cancer Center, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA

A large body of evidence has been accumulated that of the cell cycle machinery whose deregulated expres- demonstrates dominant eects of Id proteins on dierent sion might confer a proliferative advantage to tumor aspects of cellular growth. Generally, constitutive cells (Lasorella et al., 2000). Thus, one of the reasons expression of Id not only blocks cell dierentiation but why tumor cells might boost expression of Id2 is the also drives proliferation. In some settings, it is sucient demonstrated ability of high levels of Id2 to to render cells immortal or induce oncogenic transforma- functionally eliminate the Rb tumor suppressor path- tion. The participation of Id proteins in advanced human way, a process that is widely viewed as a hallmark of malignancy, where they are frequently deregulated, has cancer (Iavarone et al., 1994; Lasorella et al., 1996). been dramatically bolstered by the recent discovery that Second, the identi®cation of Id1 and Id3 as cooperat- Id exert pivotal contributions to many of the essential ing genes for tumor-mediated angiogenesis raises the alterations that collectively dictate malignant growth. intriguing possibility that recruitment of blood vessels Relentless proliferation associated with self-suciency in during human tumorigenesis might require Id activity growth signals and insensitivity to growth inhibitory (Lyden et al., 1999). In this review, we will present the signals, sustained neoangiogenesis, tissue invasiveness newest ®ndings of this rapidly expanding ®eld, which is and migration capabilities of tumor cells all share witnessing a bewildering involvement of Id in tumor- dependency on the unlimited availability of Id proteins. igenesis. We will also entertain a discussion on the It is remarkable that many of these features recapitulate implications of the role of Id proteins in human cancer those physiologically propelled by Id proteins to support and speculate on Id proteins as new therapeutic targets normal development. We propose that the participation for cancer therapy. of Id in multiple fundamental traits of cancer may be the basis for unprecedented therapeutic opportunities. Oncogene (2001) 20, 8326 ± 8333 Id and cell proliferation

Keywords: cell dierentiation; cell cycle; retinoblasto- There is overwhelming evidence favoring the view that ma protein; Id2; neuroblastoma; angiogenesis Id proteins are essential proliferative factors for a large variety of cell types (Barone et al., 1994; Hara et al., 1994; Lyden et al., 1999; Mori et al., 2000; Wang et al., Introduction 2001; Lasorella et al., 2002). Expression of Id proteins is rapidly induced by serum and high levels persist Initially identi®ed as gene products whose function was throughout S phase (Christy et al., 1991; Hara et al., the negative control of cell dierentiation, Id proteins 1994, 1997; Lasorella et al., 2000; Yates et al., 1999). A were soon recognized as molecules coordinating the number of studies have proposed that G1 progression inhibition of dierentiation with a constellation of requires the cooperative action of Id proteins on their cellular functions including proliferation and cell cycle natural targets. Among these targets, signaling through progression, migration and invasiveness, cell fate Id proteins in vivo converges upon bHLH, Ets, Pax and determination and angiogenesis (Israel et al., 1999; perhaps other transcription factors that form com- Norton, 2000; Norton et al., 1998). Id proteins impinge plexes with Id (Benezra et al., 1990; Norton, 2000; on these cellular properties through a pleiotropic Roberts et al., 2001; Yates et al., 1999). Binding to Id ability to aect multiple molecular pathways. When proteins prevents DNA binding and results in loss of deregulated, the cellular capabilities networked by Id transcriptional activity of the engaged factor. It is still proteins collectively dictate malignant transformation. unknown, however, whether inhibition of each tran- Two fundamental functions of Id proteins greatly scription factor is essential for the positive role of Id contribute to their role as potential cancer genes. First, proteins on cell proliferation. In mammalian cells, a the fact that at least one member of the Id family, Id2, crucial checkpoint control for proliferation is provided is a functional target of the retinoblastoma protein by the pocket proteins of the Rb family (Rb, p107, (Rb) suggests that Id proteins are crucial components p130) (Sherr, 1996; Weinberg, 1995). A member of the Id family, Id2, binds to each of the three pocket proteins in a cell-cycle regulated fashion and, when in *Correspondence: A Iavarone; E-mail: [email protected] large excess, inhibits their antiproliferative function Id from development to cancer A Lasorella et al 8327 (Iavarone et al., 1994; Lasorella et al., 1996, 2000). phosphorylation of Id2 and Id3 might be a mechanism What is the physiologic role of the Rb-Id2 interaction to extinguish their activity and allow progression to the for regular cell cycle progression? Two main arguments next phase of the cell cycle (Krek et al., 1994). favor a model in which Id2 operates as a downstream Because overexpression of Id proteins in tissue target of the Rb family of proteins: the rescue of culture cells results in extended proliferation, it seems embryonic lethality of Rb-null mice by ablation of Id2 likely that a similar eect will be generated in tumor and the large excess of active, hypophosphorylated cells that express abnormal amounts of Id proteins. Id pocket proteins over Id2 in normal cells (Lasorella et ablation experiments were recently performed in al., 2000). But if Rb must keep control of Id2 to neuroblastoma and in pancreatic and mammary implement its anti-proliferative function, which are the carcinoma using oligonucleotide antisense (Klee et pathways that signal following the Rb-mediated al., 1998; Lin et al., 2000; Lasorella et al., 2002). Partial inactivation of Id2? It is likely that one or more elimination of Id2 in neuroblastoma and pancreatic targets of Id2 (bHLH, Ets, Pax, etc.) are essential carcinoma and Id1 in mammary carcinoma decreased eectors of the Rb-mediated cell cycle arrest and their proliferation. In some cases reduced proliferation was activity requires restraint of Id2 function by Rb. In associated with dramatic inhibition of the malignant addition to Id2, transcription factors of the E2F family behavior of the tumor cells. These experiments lend are also essential for Rb function (Dyson, 1998). signi®cant credibility to the hypothesis that tumor cells However, the relationship between cellular Rb-E2F depend, at least in part, on Id expression for unre- and Rb-Id2 complexes is poorly understood. It is stricted proliferation and perhaps for other phenotypic possible that the same molecule of Rb will bind Id2 features of malignant transformation. and E2F. Alternatively, Id2 and E2F will compete for Many of the new capabilities acquired by malignant binding to Rb. In the ®rst scenario, in which Id2 and cells recapitulate normal characteristics of embryonal E2F are simultaneously targeted by Rb, Id2 and E2F cells (Evan and Vousden, 2001; Hanahan and Weinberg, are involved in independent functions orchestrated in 2000). This is especially true for tumors arising in combination to allow cell cycle progression. In this children. These tumors (such as neuroblastoma, medul- model, the cellular excess of pocket proteins is the loblastoma, rhabdomyosarcoma, etc.) are thought to main safeguard mechanism of negative control on cell derive from alterations of the normal processes that cycle progression. The abundant Rb-Id2 and p107-Id2 control proliferation and dierentiation during develop- complexes that are found in S phase may extinguish ment (Walterhouse and Yoon, 1997). It is therefore not the action of Id2 on its natural targets, after the surprising that the analysis of mechanisms underlying relatively unopposed Id2 activity that characterizes G1 the role of Id proteins in normal development has yielded progression. The negative role of pocket proteins on signi®cant insight into tumorigenesis. Id1, Id3 double Id2 could be essential for progression through the S null mice display defective proliferation and premature phase and cell survival. In the second model, in which dierentiation in the developing brain suggesting that the Id2 and E2F compete for Rb binding, at least some combined action of Id1 and Id3 is essential for functions of Id2 might result from the release of the proliferation of neural progenitors in vivo (Lyden et al., restraining activity of the pocket proteins on E2F 1999). Consistent with the small body phenotype transcription. The two models are not mutually associated with elimination of Id2 in the mouse, exclusive. A stoichiometric excess of Id2 over active proliferation of various cell types requires intact Id2 Rb may prevent anti-proliferative signaling from Id2 (Mori et al., 2000; Wang et al., 2001; Yokota et al., 1999; targets and, at the same time, relieve E2F transcription Lasorella et al., 2002). So far, we know that mammary from the repressive in¯uence of Rb. The recent epithelial cells, oligodendrocyte precursor cells and demonstration that Id1 inhibits Ets-mediated transcrip- embryonic ®broblasts rely on Id2 for proliferation but tion of p16Ink4a, a tumor suppressor gene operating in it is possible that Id2 is critical for proliferation of many, the same pathway as Rb, suggests that abnormal if not all, cell types. production of Id will inactivate the Rb pathway Taken together, the results discussed above imply directly (Id2 and maybe Id4) and indirectly, through that Id proteins contribute signaling pathways that are inhibition of p16Ink4a, a feature that is probably shared essential for progression of the cell division cycle. In by the four Id proteins (Ohtani et al., 2001). Another normal cells, the cell-cycle speci®c interactions between mechanism proposed to regulate the role of Id proteins pocket proteins and Id2 ensure that, like for E2F in cell cycle progression and proliferation is phosphor- transcriptional activity, the Rb family properly controls ylation (Deed et al., 1997; Hara et al., 1997; Nagata et Id2-dependent events. But when, as a consequence of al., 1995; Nagata and Todokoro, 1994). Although the oncogenic transformation, Id2 accumulates in phosphorylation sites have been identi®ed in vivo and excess over the active forms of Rb, Id2 will escape changes in Id activities, such as their binding anity the negative control of its physiologic regulator. for bHLH transcription factors, have been described Ultimately, it will be the uncontrolled ®ring of events following speci®c phosphorylation events, the biologic downstream of Id2 that likely overrides the Rb tumor consequences of Id phosphorylation remain elusive. suppressor checkpoint in human cancer (Figure 1). The Interestingly, Id2 and Id3 are phosphorylated by cyclin identi®cation of these events should oer important E-cdk2 and cyclin A-cdk2 in S phase (Deed et al., insights on the physiologic mechanisms promoting G1 1997; Hara et al., 1997). As for E2F1, cdk2-mediated progression. It should also explain why Rb must

Oncogene Id from development to cancer A Lasorella et al 8328 repress Id2 to ful®l its antiproliferative and tumor ability to constitutively activate growth signaling suppressor function. (Evan and Vousden, 2001; Hanahan and Weinberg, Ets transcription factors might be some of the 2000). One strategy to eciently accomplish both targets of deregulated expression of Id proteins in aims would be to deregulate Id protein expression. human tumors. Recent studies showed that Id proteins The consequences of ectopic expression of Id proteins bind Ets1 and Ets2 preventing DNA binding and Ets- have been analysed in a number of cell types (for mediated transcriptional activation (Yates et al., 1999). review see Norton, 2000; Norton et al., 1998). First, A relevant transcriptional target of Ets is the tumor it has been shown that overexpression of Id proteins, suppressor gene p16Ink4a and Id proteins inhibit albeit at variable levels and with notable dierences expression of p16Ink4a through the inactivation of Ets in the various cell types, enhances cell proliferation (Ohtani et al., 2001). This mechanism may be and renders cells insensitive to growth factor important during replicative senescence of primary depletion (Iavarone et al., 1994; Norton and ®broblasts, when elevation of p16Ink4a is accompanied Atherton, 1998). Second, at least two members of by loss of Id proteins (Alcorta et al., 1996; Hara et al., the Id family, Id2 and Id4, have been found to 1994, 1996). By preventing expression of p16Ink4a, confer features of transformation, such as the ability deregulated Id proteins might use an additional tool to grow in soft agar, when overexpressed in NIH 3T3 to inactivate the Rb pathway in cancer. A role for Id in ®broblasts and mammary carcinoma cells, respectively the functional inactivation of p16Ink4a received support (Lasorella et al., 2002; Beger et al., 2001). Interest- by a recent study conducted in early melanoma where ingly, recent work identi®ed Id4 as a Brca1-regulating reduced expression of p16Ink4a correlated with marked gene, whose expression decreases Brca1 and enhances elevation of Id1 (Polsky et al., 2001). tumorigenicity of breast cancer cells (Beger et al., 2001). However, the mechanism through which Id4 interferes with the upstream Brca1 regulatory path- Id as potential oncogenes way is still unknown. There are also indications that Id genes might share some of the properties of Tumor cells use various strategies to disrupt path- `immortalizing' oncogenes (Alani et al., 1999; Nickol- ways leading to terminal dierentiation, a process o et al., 2000; Norton and Atherton, 1998). When known as anaplasia. This is often associated with the expressed in primary rodent ®broblasts, Id proteins

Figure 1 The Myc-Id2-Rb pathway in development and cancer. Myc proteins are intracellular sensors of mitogenic and antimitogenic signals. When constitutively activated by genetic alterations, Myc are transformed in powerful oncoproteins. Myc bind two E-boxes in the Id2 promoter and activate transcription. During normal development of the nervous system, activity of the N-Myc-Id2 pathway is restrained by Rb. Rb binds Id2 and might prevent Id2-mediated inhibition of bHLH, Ets, Pax and maybe other transcription factors. In neuroblastoma, N-myc ampli®cation and overexpression induces accumulation of Id2 to levels exceeding active Rb. Id2 would be free to inhibit transcription of the natural targets that control cell proliferation. E, E-box

Oncogene Id from development to cancer A Lasorella et al 8329 Table 1 Deregulated expression of Id mRNAs and proteins in primary human tumors Deregulated Id Method of detection Tumor type Reference

Id1, Id3 ISHa Glioblastomab, Medulloblastomab, Neuroblastomab Lyden et al., 1999 Id1, Id2, Id3 IHCc Pancreatic cancer Maruyama et al., 1999 Id1, Id2, Id3, Id4 IHC Seminoma Sablitzky et al., 1998 Id1 IHC Medullary thyroid cancer Kebebew et al., 2000 Id1, Id2, Id3 IHC Squamous cell carcinoma Langlands et al., 2000 Id1 IHC Breast carcinoma Lin et al., 2000 Id1 IHC Endometrial cancer Takai et al., 2001 Id1 IHC Cervical cancer Schindl et al., 2001 Id1 ISH Melanoma Polsky et al., 2001 Id2 IHC Neuroblastoma Lasorella et al., 2002 aIn situ hybridization; bld mRNAs are expressed in blood vessels; cImmunohistochemistry generate `immortal' foci and display a cooperative targets as well as pre-clinical testing of new anti-Id eect when co-expressed with ras (Norton and drugs. Atherton, 1998). It was also reported that constitutive expression of Id1 could immortalize primary keratinocytes (Alani et al., 1999). However, a more recent Id in development and cancer of the nervous system study in which Id1 expression in keratinocytes was driven by retroviral vectors in the absence of drug A widely accepted assumption postulates that Id selection showed that expression of Id1 could extend proteins are abundant in proliferating, undierentiated the life span but was not sucient for immortaliza- cells but low or undetectable levels of Id are present in tion (Nickolo et al., 2000). Probably built as an post-mitotic, dierentiated and senescent cells (Norton, important limiting factor to the growth promoting 2000). Probably, this can still be considered the rule. capacity, the pro-apoptotic ability of Id proteins However, `exceptions' have now been described for a might provide a counterforce to prevent full number of cell types in which high levels of Id proteins immortalization (Florio et al., 1998; Kee et al., were found in dierentiated cells that have exited the 2001; Norton and Atherton, 1998). Thus, it was not cell cycle (Chen et al., 1999; Cooper et al., 1997; surprising that inhibition of apoptosis by members of Ishiguro et al., 1996; Jen et al., 1997; Neuman et al., the Bcl-2/Bcl-x antiapoptotic family greatly enhanced 1993). This behavior typically concerns Id2 and Id4 Id-mediated immortalization. By sharing the dual that are expressed by a variety of dierentiating cell ability to stimulate cell proliferation and cell death, types from the neuroectodermal lineage. Whether or Id proteins join the group of bona ®de growth- not Id have tissue-speci®c functions in these cell types promoting proteins. For example, Myc and E2F1 are is unknown. A report indicated that Id2, when powerful activators of apoptosis and, at least in the overexpressed, determines cell fate speci®cation in the case of Myc, the oncogenic action is strongly ectoderm by driving cells along the neural crest potentiated by survival genes of the Bcl-2/Bcl-x phenotype while suppressing the epidermal lineage family (Dyson, 1998; Grandori et al., 2000). (Martinsen and Bronner-Fraser, 1998). On the other Probably the strongest evidences developed so far to hand, the abnormal overgrowth of neural crest clusters justify the inclusion of Id in the group of nuclear in this system is consistent with a proliferative oncogenes are three independent studies where trans- advantage conferred to these cells by overexpression genic mouse lines were engineered to express Id1 and of Id2. Id2 in the lymphoid system and Id1 in the small A possible explanation for the expression of Id in intestine (Kim et al., 1999; Morrow et al., 1999; Wice dierentiating cells could be to ensure the persistence and Gordon, 1998). Targeted expression of Id1 and Id2 of a temporarily inactive proliferative machinery ready in T cells generated aggressive thymic lymphoma which to resume activity under special circumstances (Lasor- rapidly killed the mouse (Kim et al., 1999; Morrow et ella et al., 2000). In normal conditions, Id proteins in al., 1999). As a further indication of the broad post-mitotic cells would be kept under strict negative phenotypic spectrum that may arise from Id activation, control by upstream regulators. This model is illus- expression of Id1 in intestinal epithelial cells generated trated by the control of Rb upon Id2, a critical seemingly benign intestinal adenomas (Wice and mechanism to prevent inappropriate Id2 signaling in Gordon, 1998). dierentiating neurons (Lasorella et al., 2000). In the Because deregulation of Id proteins is spread among absence of Rb, neurons undergo ectopic proliferation multiple types of human cancer (Table 1), there is and apoptosis (Lee et al., 1994; Slack et al., 1998). compelling pressure to generate murine models where Interestingly, these defects are rescued by ablation of transgenic Id expression in dierent cell lineages could Id2 in vivo and are recapitulated in vitro by over- recapitulate speci®c biologic features of human expression of Id2 in normal cortical progenitors malignancies. Such models would provide invaluable (Lasorella et al., 2000; Toma et al., 2000). By inhibiting systems for the functional screening of candidate Id Id2 function, Rb has been proposed to collaborate with

Oncogene Id from development to cancer A Lasorella et al 8330 neural-speci®c bHLH transcription factors during functionally restore tumor angiogenesis in Id mutant neurogenesis to ensure exit from cell cycle, terminal mice (Lyden et al., 2001). Accordingly, disruption of dierentiation and survival. Remarkably, oncogenic Id1 and Id3 appears to result in the failure to mobilize transformation in the nervous system takes advantage bone marrow precursors that are essential for the of deregulated expression of Id2 to permanently neovascular postnatal process. Thus, VEGF-induced inactivate the growth inhibitory function of Rb (see activation of Id1 and Id3 may be a critical molecular below). event required to promote mobilization of bone It is possible that other negative regulators of Id marrow precursors. In conclusion, there is now function will be discovered in the near future. A recent convincing evidence for a cooperative action of Id1 study proposed that dierentiation of oligodendrocyte and Id3 in neoangiogenesis, a vital process for precursor cells requires nuclear exclusion of Id2 (Wang continuous growth of tumor cells. It is likely that et al., 2001). It will be interesting to test whether signaling pathways initiated by tumor cells and regulation of subcellular location will be of general involved in the recruitment of neighboring endothelium importance in other cell lineages and whether it also and in the mobilization of bone marrow precursors pertains to other members of the Id family. Nuclear trigger multiple Id gene responses in the target cells. exclusion of Id proteins may be accomplished through The detection of abnormally high expression of Id1 a dominant mechanism, for example by cytoplasmic and Id3 in endothelial cells in®ltrating human neuro- proteins sequestering Id. Identi®cation of these factors blastoma, medulloblastoma and glioblastoma multi- should oer new tools for the ongoing eorts aimed at forme (Lyden et al., 1999) raises the possibility that extinguishing Id function in tumor cells carrying spontaneous human tumors require signaling converg- deregulated expression of Id. ing on Id proteins for neovascularization.

Id proteins in tumor angiogenesis and tissue invasion Deregulation of Id in primary tumors

In the whole organism, two novel capabilities must be Numerous recent in situ hybridization and immuno- imposed upon mammalian cells to fully manifest the histochemical studies reported on the dysregulated transformed phenotype: the ability to produce matrix- expression of Id proteins in a large variety of human degrading proteases and the ability to promote growth tumors (Table 1). Additionally, Id protein expression of new blood vessels (Hanahan and Weinberg, 2000). has been proposed as potential prognostic factor for The ®rst is used by tumor cells to facilitate invasiveness breast and cervical cancer as well as neuroblastoma into neighboring tissues, the second is essential to (Lin et al., 2000; Schindl et al., 2001; Lasorella et al., supply oxygen and nutrients for the expanding tumor 2002). Elevation of Id1 correlates with a more mass. It is remarkable that both of these capabilities aggressive subset of breast cancer and is an indepen- appear to require the participation of Id proteins. dent marker for tumor progression in cervical cancer. In studies conducted in mammary epithelial cells, Id2 overexpression, on the other hand, is strongly forced expression of Id1 not only prevented dierentia- predictive of poor outcome in children with neuro- tion and increased proliferation but also conferred the blastoma, irrespective of other clinical and biological ability to migrate and invade the basement membrane variables. Thus, the immunohistochemical analysis of to otherwise non-tumorigenic cells (Desprez et al., Id protein expression has considerable potential to 1995, 1998). This phenotype appears to be related to enter in the practical use for the routine assessment of the production of a novel metalloproteinase of 120 kD cancer patients. However, alterations of Id genes have by Id1-expressing cells. Thus, it was not unexpected to never been described in human cancer. This simple fact ®nd that the most aggressive breast cancer cell lines prevents us from attaching the classical label of and primary tumors showed deregulated expression of `oncogenes' to Id. It is possible that the lack of genetic Id1 (Lin et al., 2000). lesions of Id re¯ects the participation in oncogenic Probably one of the most exciting functions recently networks initiated by genetic alterations of upstream attributed to Id proteins is their role in the angiogen- regulators. An example of this interplay is the esis of malignant tumors. This property was ®rst constitutive activation of the N-Myc-Id2 pathway in described in a study reporting the generation of neuroblastoma, a highly malignant embryonal tumor Id17/7, Id3+/7 double mutant mice (Lyden et al., derived from the neural crest (Lasorella et al., 2000; 1999). Remarkably, these mice failed to support the Maris and Matthay, 1999). This pathway operates growth of tumors and metastasis from a variety of through the primary ampli®cation and/or overexpres- xenografts and cancer-predisposing mutations due to sion of the N-myc oncogene. Elevation of Id2 in this poor vascularization with consequent necrosis of the tumor is a secondary event to the accumulation of N- tumor cells. Lack of MMP2 metalloproteinase activa- Myc transcription factors that directly bind to the Id2 tion and downregulation of integrins in tumor blood promoter and activate its abnormal transcription. The vessels of Id mutant mice may provide an explanation ultimate outcome is the accumulation of the Id2 for the angiogenic defect. However, recent data protein at levels that are sucient to inactivate the demonstrated that transplantation of wild-type bone Rb pathway. Thus, neuroblastoma represents the ®rst marrow-derived cells expressing VEGF receptors example of a tumor where an Rb target (Id2) is

Oncogene Id from development to cancer A Lasorella et al 8331 recruited by a powerful oncogene (N-myc) to overcome targeting the abnormal Id signaling in cancer shows the Rb tumor suppressor pathway (Figure 1). exceptionally promising potentialities. First, by hitting Other studies have identi®ed Id proteins as critical Id in tumor cells, these molecules might reconstitute downstream targets of proliferative and antiprolifera- the integrity of the Rb pathway, the most critical tive signaling pathways often perturbed in cancer. antiproliferative safeguard available to mammalian Mitogenic signals driven by estrogens, insulin growth cells. They could also prevent invasiveness by limiting factor-2 and T-cell receptor induce expression of Id1, production of matrix metalloproteinases. Second, by Id2 and Id3, respectively (Bain et al., 2001; Belletti hitting Id in the tumor vasculature, they could reduce et al., 2001; Lin et al., 2000). Recent data proposed oxygen and nutrients' supply to tumor cells. Neuro- that the extracellular signals that elevate the blastoma is the ®rst human tumor showing such a dual expression of Id might be mediated by the Ras- involvement of Id proteins. In this embryonal cancer, Erk-Map kinase cascade and the PI3 kinase pathway abnormal expression of Id2 in the tumor cells is (Bain et al., 2001; Belletti et al., 2001). On the other associated with deregulated expression of Id1 and Id3 hand, many antiproliferative signals are funneled in the tumor blood vessels (Table 1). Resistance to through the down-regulation of Id gene expression. conventional treatment and poor prognosis character- Prominent among the antimitogenic pathways that ize almost invariably advanced neuroblastoma (clinical lead to decreased expression of Id proteins are those stage 4), (Katzenstein and Cohn, 1998). We suggest initiated by progesterone and TGFb (Chen et al., that stage 4 neuroblastoma will be an excellent cancer 2001; Lasorella et al., 2000; Lin et al., 2000). model to test the ecacy of novel anti-Id drugs when Because constitutive activation of proliferative signals these substances will become available. Among poten- and disruption of antiproliferative circuits coexist in tial tumor targets for intervention, few other molecules a variety of ways in dierent types of human oer the attractive targeting mechanisms against tumors, it will not be surprising if alterations of multiple essential traits of cancer that might be Id proteins will be described in most, if not all associated with the ablation of Id function in human human malignancies. malignancy. It will be the privilege of those dedicated to this exciting ®eld of research to attempt to render these promising hopes into reality. Future perspectives

At least four essential hallmark capabilities of malig- Acknowledgments nancy are conferred by dysregulated expression of Id In preparing this review, we relied in part upon compre- proteins during tumorigenesis: anaplasia, uncontrolled hensivereviewscitedinthetextandapologizeforthe proliferation, sustained neoangiogenesis and invasion omission of many important original references. Work in of neighboring tissues (Figure 2). The design of drugs our laboratory is supported by NIH grant RO1-CA85628.

Figure 2 Id signaling controls multiple essential traits of cancer. Id proteins inactivate bHLH transcription factors and inhibit dierentiation. They also disrupt antiproliferative pathways and promote proliferation. By acting as mediators of VEGF signaling, Id allow mobilization of angiogenic precursors and recruitment of neighboring endothelium. Id also stimulate production of metalloproteinases to facilitate tissue invasion. Unrestricted Id activity in human cancer suppresses terminal dierentiation (anaplasia), causes relentless proliferation, induces angiogenesis and promotes tissue invasiveness

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