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(2006) 25, 5233–5243 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc REVIEW The tumor-suppressor , the exception that proves the rule

DW Goodrich

Department of Pharmacology & Therapeutics, Roswell Park Institute, Buffalo, NY, USA

The retinoblastoma tumor-suppressor gene (Rb1)is transmission of one mutationally inactivated Rb1 centrally important in . Mutational and loss of the remaining wild-type allele in somatic inactivation of Rb1 causes the pediatric cancer retino- retinal cells. Hence hereditary retinoblastoma typically , while deregulation ofthe pathway in which it has an earlier onset and a greater number of tumor foci functions is common in most types of human cancer. The than sporadic retinoblastoma where both Rb1 Rb1-encoded protein (pRb) is well known as a general must be inactivated in somatic retinal cells. To this day, cycle regulator, and this activity is critical for pRb- Rb1 remains an exception among cancer-associated mediated tumor suppression. The main focus of this in that its is apparently both necessary review, however, is on more recent evidence demonstrating and sufficient, or at least rate limiting, for the genesis of the existence ofadditional, cell type-specific pRb func- a human cancer. The simple of retinoblastoma tions in cellular differentiation and survival. These has spawned the hope that a complete molecular additional functions are relevant to sug- understanding of the Rb1-encoded protein (pRb) would gesting that the net effect of Rb1 loss on the behavior of lead to deeper insight into the processes of neoplastic resulting tumors is highly dependent on biological context. transformation and, ultimately, to therapeutic benefit The molecular mechanisms underlying pRb functions are for patients. This hope has been buoyed by the discovery based on the cellular proteins it interacts with and the that Rb1 mutation is observed in a number of other functional consequences of those interactions. Better common human . The expression of proteins that insight into pRb-mediated tumor suppression and clinical regulate pRb are also frequently altered in an even exploitation ofpRb as a therapeutic target will require a broader spectrum of cancers, indicating that deregula- global view ofthe complex, interdependent network of tion of the normal pathways in which pRb functions pocket protein complexes that function simultaneously may be very common in human cancer (Sherr, 2000; within given tissues. Malumbres and Barbacid, 2001). What is learned about Oncogene (2006) 25, 5233–5243. doi:10.1038/sj.onc.1209616 pRb, therefore, is likely to be applicable to cancer in general. Keywords: tumor-suppressor gene; ; differen- Shortly after its identification, pRb was identified as tiation; DNA damage repair; a binding partner for mitogenic oncoproteins expressed by DNA tumor including SV40 large T antigen (DeCaprio et al., 1988), adenovirus E1A (Whyte et al., 1988), and papillomavirus E7 proteins (Munger et al., 1989). In addition, pRb was discovered to be phos- The retinoblastoma tumor-suppressor gene (Rb1) has phorylated in synchrony with the cell cycle (Buchkovich been a focal point for research into the molecular et al., 1989; Chen et al., 1989; DeCaprio et al., 1989). underpinnings of cancer since its identification and This data suggested that pRb may be a general cell cycle cloning nearly 20 years ago (Dryja et al., 1986; Friend regulator. This hypothesis was initially supported by the et al., 1986; Fung et al., 1987; Lee et al., 1987). As observations that exogenous unphosphorylated pRb foreshadowed by Knudson’s ‘two-hit hypothesis’ for the could arrest cells in the of the cell cycle origins of retinoblastoma (Knudson, 1971), mutational (Goodrich et al., 1991; Connell-Crowley et al., 1997), inactivation of both Rb1 alleles is the primary molecular and that depletion of pRb lead to an accelerated G1/S alteration causing the pediatric cancer retinoblastoma. transition (Herrera et al., 1996). An important role In about 10% of retinoblastoma cases, the disease is for pRb in cell cycle regulation has been consistently inherited as a simple autosomal dominant trait with validated by research over the ensuing decade. The greater than 95% (Lohmann and Gallie, reader is referred to a recent review appearing in the 2004). Hereditary retinoblastoma is caused by pages of this journal for a more in depth review of this topic (Cobrinik, 2005). The finding that pRb could bind viral proteins Correspondence: Dr DW Goodrich, Department of Pharmacology & Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, spurred the search for cellular proteins that might Buffalo, NY 14263, USA. interact with pRb. The first cellular pRb-binding partner E-mail: [email protected] discovered was the factor (Helin Rb1 functions unrelated to the cell cycle DW Goodrich 5234 et al., 1992; Kaelin Jr. et al., 1992; Shan et al., 1992). The Rb1 protein has also been implicated in other aspects active, unphosphorylated form of pRb preferentially of cell cycle control. For example, pRb can inhibit bound E2F1, and disruption of the pRb/E2F1 complex progression by attenuating cyclin A/Cdk2 caused cell cycle deregulation (Shan et al., 1996). It was activity, resulting in disruption of PCNA function and soon realized that E2F1 is a member of a family of DNA replication (Knudsen et al., 1998; Sever-Chroneos related sequence-specific DNA-binding transcription et al., 2001). Rb1 protein is required for efficient G1/S factors. Rb1 protein was able to bind several members cell cycle checkpoint activation in response to DNA of this family, particularly the transcriptional activators damage (Harrington et al., 1998), and is required to E2F1–3. When in complex with E2F1–3, pRb blocked maintain cell cycle exit in quiescent and senescent cells their ability to activate transcription. In addition, pRb (Sage et al., 2003). Loss of pRb can cause G2/M cell recruited a number of modifying factors, cycle defects through deregulation of the target such as deacetylases, to facilitate active gene gene Mad2, a mitotic checkpoint gene (Hernando et al., silencing. As E2F activity regulated many cell cycle 2004). Rb1 protein can also regulate the cell cycle genes (Ishida et al., 2001; Ren et al., 2002) and was through mechanisms that are independent of E2F. For required for a normal cell cycle (Humbert et al., 2000; example, pRb can interact with Skp2, the substrate- Wu et al., 2001), repression of E2F-dependent transcrip- recognition subunit of the protein ligase tion has generally been considered the principal SCFSKP2. Binding of pRb to Skp2 prevents degradation mechanism underlying pRb-mediated cell cycle control. of the CDK inhibitor p27KIP1, a normal target for This hypothesis was supported by in vivo evidence SCFSKP2-mediated protein degradation (Ji et al., 2004). indicating that loss of Rb1 lead to unscheduled cell Stabilization of p27KIP1 inhibits the CDK activity that is proliferation in mice, and that compound loss of required for normal cell cycle progression. Likewise, individual E2F family members partially rescued this pRb can bind the Mitf and cell cycle defect (Tsai et al., 1998; Ziebold et al., 2001; cooperate with Mitf in activating transcription of the Saavedra et al., 2002). Dimova and Dyson (2005) CDK inhibitor p21CIP1, at least in some cell types recently reviewed the scientific literature relevant to the (Carreira et al., 2005). E2F family of transcription factors. Despite the significant progress that has been made in The ability of pRb to bind E2Fs, silence E2F- elucidating the general role of pRb in cell cycle control, dependent transcription, and restrain cell cycle progres- it is still not entirely clear what contribution these sion is inhibited by phosphorylation. The activity of mechanisms make to pRb-mediated tumor suppression. cyclin-dependent kinases (CDKs) correlates with the While it makes intuitive sense that pRb suppresses onset of pRb phosphorylation and the G1/S cell cycle tumorigenesis by restraining the cell cycle, some have transition. Cyclin-dependent kinases can phosphorylate argued that cancer is fundamentally a disease of pRb in vitro and this phosphorylation inhibits pRb/E2F defective differentiation (Harris, 2004). Perhaps pRb complex formation (Dynlacht et al., 1994), as well as pRb- integrates the processes of cell cycle exit and cellular mediated cell cycle arrest activity (Connell-Crowley differentiation. Indeed, recent evidence indicates pRb et al., 1997). Regulation of pRb by phosphorylation is has additional functions that may have a direct role in complex. Up to 16 possible CDK phosphorylation sites the regulation of cellular differentiation and cell exist on pRb, and multiple CDKs can phosphorylate survival. These additional functions are likely to pRb with some site specificity (Connell-Crowley et al., influence the behavior of cells that lose pRb activity. 1997; Zarkowska and Mittnacht, 1997; Takaki et al., 2005). Other proline-directed kinases like p38 stress activated kinase 1 (Hou et al., 2002; Nath et al., 2003), ERK1/2 (Guo et al., 2005), and possibly Raf-1 The pocket protein family (Dasgupta et al., 2004) can also phosphorylate pRb. Whereas some phosphorylation sites appear to be One of the factors that complicates attempts to unravel critical for negative regulation of pRb (Knudsen and the molecular mechanisms responsible for pRb- Wang, 1996; Connell-Crowley et al., 1997), the effects of mediated tumor suppression is the presence of two phosphorylation at individual sites varies depending on cellular proteins, p107 and p130 that are structurally the phosphorylation status of other sites (Knudsen and related to pRb. All three proteins contain a protein Wang, 1997; Brown et al., 1999; Barrientes et al., 2000). interaction domain called the ‘pocket’ that is required to How phosphorylation at individual sites or combina- mediate their interactions with other viral and cellular tions of sites regulates pRb function has not been clearly proteins. All of the pocket proteins share the ability to defined. A model emerges wherein pRb functions as a bind E2Fs, restrain cell cycle progression, and serve as general cell cycle inhibitor that restrains the G1/S substrates for CDKs (Cobrinik, 2005). This functional transition by binding E2F and repressing E2F-depen- overlapis reflected in the observation that cells lacking dent transcription. Phosphorylation of pRb, as a result p107 or p130 have cell cycle defects similar to those of appropriate mitogenic stimulation and activation of lacking pRb (Hurford et al., 1997; Classon et al., 2000b), CDKs, blocks pRb/E2F complex formation resulting in and loss of all three pocket proteins causes more derepression and activation of E2F-dependent gene pronounced cell cycle defects than loss of individual expression. This E2F-dependent facil- members (Dannenberg et al., 2000; Sage et al., 2000). itates continued cell cycle progression. Functional compensation is also observed in vivo as

Oncogene Rb1 functions unrelated to the cell cycle DW Goodrich 5235 developmental defects associated with Rb1 loss are Rb1 and cellular differentiation exacerbated by compound loss of p107 (Lee et al., 1996a; Chen et al., 2004; MacPherson et al., 2004; The advent of mice with genetically engineered deletions Zhang et al., 2004b) or p130 (MacLellan et al., 2005). of the Rb1 gene has led to a growing appreciation of the Despite the existence of functional compensation important role it plays in cell fate specification and between the pocket proteins, it is clear that pRb has differentiation. Rb1 null mice die in mid-gestation unique functions. Only pRb among the pocket proteins (Clarke et al., 1992; Jacks et al., 1992; Lee et al., is required for embryonic development (Clarke et al., 1992). Defects are observed in a number of tissues 1992; Jacks et al., 1992; Lee et al., 1992) whereas mice including the eye lens, brain, peripheral nervous system, lacking p107 or p130 develop normally (Cobrinik et al., muscle, liver, placenta, and the hematopoietic system 1996; Lee et al., 1996b). The molecular bases for these among others (Novitch et al., 1996; Zacksenhaus et al., differences are not entirely clear. For one, the pocket 1996; Tsai et al., 1998; Wu et al., 2003; Iavarone et al., proteins are expressed at different times in the cell cycle 2004; Spike et al., 2004). Conditional, tissue-specific and are likely to have different, but overlapping, gene ablation has also been used to examine the effects expression patterns in vivo. Moreover, pRb preferen- of Rb1 loss in adult tissue. Such studies have implicated tially binds E2F1–4 while p107 and p130 primarily Rb1 in the normal development of the epidermis interact with –5. This probably accounts for the (Balsitis et al., 2003; Ruiz et al., 2004), melanocytes observation that while loss of individual pocket proteins (Yu et al., 2003), hair cells (Sage et al., 2005), liver has similar effects on the cell cycle, the E2F target genes (Mayhew et al., 2005), prostate (Maddison et al., 2004), deregulated in each case are different (Hurford et al., lung (Wikenheiser-Brokamp, 2004), cerebellum (Marino 1997; Wells et al., 2000). However, it should be noted et al., 2003), pituitary (Vooijs et al., 1998), and retina that even p107 and p130 can bind and regulate E2F1–3 (Chen et al., 2004; MacPherson et al., 2004; Zhang et al., under certain conditions (Lee et al., 2002). 2004a). Affected tissues typically show signs of un- Perhaps the most important difference between the scheduled , incomplete differentiation, pocket proteins is that only mutation of Rb1 is and apoptotic cell death. Although some of these tissues consistently associated with tumorigenesis in both mice initiate a differentiation program in the absence of pRb, and humans. Heterozygous Rb1 null mice develop as indicated by the expression of early lineage-specific normally, but spontaneously develop pituitary tumors markers, they generally fail to reach a completely of the intermediate and anterior lobes as well as differentiated state. medullary thryroid with nearly complete The developmental defects observed upon loss of Rb1 penetrance (Hu et al., 1994; Zhou et al., 2005). have been attributed to both cell autonomous and non- Conditional ablation of Rb1 in mouse skin (Balsitis cell autonomous mechanisms. For example, the failure et al., 2003; Ruiz et al., 2004) or prostate (Maddison of proper placental development (Wu et al., 2003) and/ et al., 2004) also leads to the appearance of hyperplastic or fetal liver macrophage differentiation (Iavarone et al., lesions. Conditional ablation of pRb in the mouse 2004) contributes to the failure of erythrocyte matura- cerebellum in the absence of can cause medullo- tion in the absence of Rb1. Rb1À/À erythroblasts also fail blastoma (Marino et al., 2000), and loss of pRb and to differentiate normally in vitro or when reconstituting p107 is sufficient for the genesis of murine retinoblast- the hematopoietic system in irradiated wild-type mice oma (Chen et al., 2004; MacPherson et al., 2004; Zhang (Clark et al., 2004; Spike et al., 2004). Thus, the pRb- et al., 2004a). In contrast, mice completely lacking p107 associated hematopoiesis defects are due to both non- or p130 are not tumor prone. In humans, mutational cell autonomous and cell autonomous mechanisms. In inactivation of Rb1 is a prerequisite for retinoblastoma. turn, the hematopoiesis defect contributes to develop- Rb1 mutation also occurs in a large fraction of mental defects observed in the central nervous system osteosarcomas and soft tissue and these (MacPherson et al., 2003). Conditional ablation of Rb1 diseases have been epidemiologically linked to retino- in the central nervous system yields only a subset of the blastoma (Bookstein and Lee, 1991). Rb1 mutation is brain observed in Rb1 nullizygous mice also detected in almost all small-cell lung carcinomas because hematopoiesis is not compromised in the and, to a lesser extent, in of the breast, conditionally mutant mice. Rb1 loss, therefore, elicits prostate, and bladder to name a few. Consistent with the a number of direct and indirect effects on cellular data in mice, mutational inactivation of p107 or p130 is differentiation and survival that contribute to the rarely observed in human cancer. Although arguments developmental phenotypes observed. have been made in support of potential tumor-suppres- In vitro differentiation model systems have also sor activity for p130 and p107 in human cancer (Paggi provided support for a direct role for pRb in cellular and Giordano, 2001), it is clear that among pocket differentiation. Loss of pRb compromises, and exogen- proteins, pRb plays a special role in tumor suppression. ous pRb expression restores muscle differentiation Thus, the identification of molecular mechanisms in vitro (Gu et al., 1993; Schneider et al., 1994). Initial unique to pRb are likely to be particularly relevant growth arrest and expression of early differentiation to carcinogenesis. However, in assessing the possible markers does not require pRb, but permanent cell cycle consequences of pRb inactivation, it is important to withdrawal and expression of late differentiation mar- consider the potentially overlapping functions of the kers does (Schneider et al., 1994; Novitch et al., 1996). other pocket proteins. In contrast cells lacking p107 and/or p130 are fully

Oncogene Rb1 functions unrelated to the cell cycle DW Goodrich 5236 capable of muscle differentiation in vitro. Rb1 is also grams. The ability of pRb to bind and augment the required for adipocyte differentiation (Chen et al., transcriptional activity of tissue-specific transcription 1996a) and lineage commitment (Hansen et al., 2004) factors like MyoD (Gu et al., 1993), C/EBPs (Chen in vitro. Emphasizing the unique role pRb plays in this et al., 1996a), NF-IL6 (Chen et al., 1996b), Pax8 system, loss of p107 and p130 actually facilitates (Miccadei et al., 2005), CBFA1 (Thomas et al., 2001), adipogenesis (Classon et al., 2000a). Consistent with and nuclear receptors (Singh et al., 1995; Lu the frequent mutation of Rb1 observed in osteosarcoma, and Danielsen, 1998; Batsche et al., 2005) to name a few pRb also potentiates osteogenic differentiation in vitro supports the latter hypothesis. However, there is some (Thomas et al., 2001). debate whether a pRb/MyoD complex is directly As cellular differentiation is tightly coupled to cell responsible for augmentation of muscle-specific gene cycle exit, a major unanswered question is whether expression (Li et al., 2000). An alternative explanation is the effects of pRb on differentiation reflect additional, that pRb can bind and block the activity of differentia- cell type-specific mechanisms, or whether they are tion inhibitors like EID-1 (MacLellan et al., 2000; an indirect consequence of pRb-mediated cell cycle Miyake et al., 2000), RBP2 (Benevolenskaya et al., control (Figure 1). In other words, does pRb facilitate 2005), or Id2 (Iavarone et al., 1994, 2004). These differentiation simply by enforcing cell cycle exit, or inhibitors block the ability of lineage-specific transcrip- does it use additional mechanisms to directly facilitate tion factors to activate gene expression. Hence pRb the execution of lineage-specific gene expression pro- may augment lineage-specific gene expression indirectly by blocking the activity of inhibitors, or directly by augmenting the activity of tissue-specific transcriptional activators. There is accumulating evidence that pRb-mediated mechanisms involved in cellular differentiation are separable from the ability of pRb to restrain the cell cycle. For example, increased expression of EID-1 blocks the ability of pRb to activate lineage-specific gene expression, but does not block the ability of pRb to repress transcription and constrain the cell cycle (MacLellan et al., 2000; Krutzfeldt et al., 2005). Compound loss of Rb1 and N- or K-Ras in mice rescues a subset of phenotypes characteristic of Rb1 nullizygous mice. In particular, defective differentia- tion is improved upon compound loss of a Ras gene despite evidence that the deregulated cell proliferation typical of Rb1 nullizygous embryos is unaffected (Takahashi et al., 2003, 2004). Evidence from Rb1 null mouse retinae indicates there is only mild deregulation of retinal progenitor cell proliferation but a dramatic reduction in mature rod photoreceptors (Sage et al., 2003; Donovan and Dyer, 2004; MacPherson et al., 2004; Zhang et al., 2004a; Dyer and Bremner, 2005; Schweers and Dyer, 2005). Lineage and gene expression analysis suggests that the role of pRb in rod photo- Figure 1 Models for explaining the effects of pRb on cellular differentiation is distinct from its role in retinal differentiation and survival. During development, pRb is a wide progenitor cell proliferation (Zhang et al., 2004a). spread regulator of the cell cycle. However, loss of pRb elicits tissue-specific defects in cellular differentiation and survival as well Perhaps the most compelling evidence that pRb as in cellular proliferation. The effects of pRb loss on differentia- utilizes cell cycle independent mechanisms to regulate tion and survival may be an indirect consequence of the inability to cellular differentiation is the identification of pRb exit the cell cycle (a). This model is based on the frequent mutants that genetically separate its effects on differ- observation that cellular differentiation and apoptotis is coupled to the cell cycle. The model predicts that the molecular mechanisms entiation from its effects on the cell cycle. Several Rb1 underlying Rb1-associated differentiation and survival defects will have been identified that fail to stably bind be similar to those involved in pRb-mediated cell cycle control, E2F transcription factors, fail to repress E2F-dependent primarily negative regulation of E2F transcription factors. Rb1- transcription, and fail to regulate the cell cycle. Yet, dependent differentiation and survival may also be mediated by these mutants are still able to activate lineage-specific direct mechanisms (b). In this model, pRb is proposed to utilize additional, cell cycle independent mechanisms to directly influence gene expression and facilitate cellular differentiation cell differentiation and survival. These mechanisms may or may not in vitro (Sellers et al., 1998). In some cases, the ability of involve E2F transcription factors. This model predicts that the these mutants to promote differentiation correlates with effects of pRb on proliferation, differentiation, and apoptosis their ability to bind proteins like RBP2 (Benevolenskaya should be genetically separable, at least in some cases. The models are not mutually exclusive. It is conceivable that both models are et al., 2005), thus underscoring the potential biological operable, and the relative contribution of each to the phenotypes relevance of these protein interactions. One of these associated with pRb loss may be dependent on biological context. pRb mutants, an arginine for tryptophan substitution at

Oncogene Rb1 functions unrelated to the cell cycle DW Goodrich 5237 codon 661, is naturally occurring and is associated with although this apoptosis is mediated through E2F1 partially penetrant hereditary retinoblastoma (Onadim because the E2F1 promoter is itself a target for et al., 1992; Lohmann et al., 1994). This mutation regulation by activating E2Fs (Denchi and Helin, belongs to a small class of naturally occurring, partially 2005). Gene expression profiling has suggested that penetrant Rb1 mutations that affect the primary E2F1 regulates genes important for apoptosis (Muller structure of pRb, but do not affect Rb1 expression. et al., 2001), including the apoptosis protease activating Presumably the residual differentiation promoting factor-1 gene (Apaf1) and procaspases (Moroni et al., functions of the mutant protein account for the reduced 2001; Nahle et al., 2002). Apaf1 is a key downstream penetrance and of retinoblastoma observed effector of the mitochondrial pathway of apoptosis and in families carrying the mutant allele (Harbour, 2001). is upregulated in the absence of pRb, presumably due to Our laboratory has recently generated the analogous the effects of E2F1 deregulation. Procaspase activation mutation (R654W) in mice in order to assess the relative is the rate-limiting step in apoptotic cell death, so higher contribution of pRb/E2F-depedent and -independent levels of procaspases presumably sensitize cells to mechanisms to embryonic development. Like Rb1 apoptosis. Compound loss of Apaf1 or procaspases-3 nullizygous mice, mice homozygous for this mutant diminishes the extent of apoptosis observed in the allele exhibit widespread deregulation of the cell cycle as central nervous system of Rb1 nullizygous embryos might be expected given its inability to stably bind and (Guo et al., 2001; Simpson et al., 2001). regulate the transcriptional activators E2F-1, -2, and -3. While pRb may influence both the cell cycle and cell Despite this, homozygous mutant embryos live longer survival through interaction with E2F transcription than Rb1 nullizygous embryos, and exhibit improved factors, it is not clear how expression of the cell cellular differentiation in some tissues (Sun et al., cycle and apoptotic transcriptional targets of E2F are 2005). For example, improved erythrocyte maturation coordinated to determine the appropriate cell fate. A and fetal liver macrophage differentiation is observed paradox arises wherein a cell that is fated to survive and compared to Rb1 nullizygous tissue. Improved differ- proliferate will contain phosphorylated pRb in order to entiation correlates with the ability of the R654W pRb disrupt pRb/E2F complexes and relieve restraint on the to retain protein interactions with E2F4 and Id2, two cell cycle. However, disruption of pRb/E2F complexes is transcription factors known to influence the differentia- expected to upregulate both cell cycle and apoptotic tion of these tissues. Thus, the mechanisms that pRb E2F target genes. Conversely, it is paradoxical for a uses to facilitate cellular differentiation in these tissues is tumor-suppressor gene to inhibit apoptosis given that genetically separable from its general ability to stably this is a well-known oncogenic mechanism. This bind activating E2F transcription factors and regulate functional property predicts that pRb expression may the cell cycle. be oncogenic, a prediction that has actually been verified under certain experimental conditions (Yamamoto et al., 1999; Jiang and Zacksenhaus, 2002; Borges Rb1 and cell survival et al., 2005). Hence the cell must have some way to suppress the proapoptotic effects of pRb inactivation as A prominent observed in Rb1 null embryos is it attempts to proliferate. One possibility is the presence excessive apoptosis in several tissues including the of a distinct, E2F1-specific binding site on the carboxy- nervous system, eye lens, and skeletal muscle (Clarke terminus of pRb that may be important for regulation of et al., 1992; Jacks et al., 1992; Lee et al., 1992). Ectopic apoptosis, but not cell proliferation (Dick and Dyson, expression of pRb has also been demonstrated to confer 2003). Presumably, a distinct pRb/E2F1 complex that resistance to a number of apoptotic triggers in vitro suppresses apoptosis would be retained even after pRb (McConkey et al., 1996). As with cellular differentiation, phosphorylation relieves cell cycle restraint. Similarly, the analogous question arises whether the ability to phosphorylated pRb has been proposed to interact with regulate apoptosis is an indirect consequence of pRb- and inhibit the proapoptotic nuclear protein pp32 mediated cell cycle regulation, or whether the apoptotic (Adegbola and Pasternack, 2005). Alternatively, the cell defects reflect distinct pRb mechanisms that directly may utilize independent antiapoptotic pathways, per- regulate apoptosis (Figure 1). Consistent with the haps mediated by survival factors, to dampen the former explanation, excessive apoptosis is typically expected proapoptotic effects of pRb/E2F complex observed in the same tissues that exhibit loss of cell disruption. cycle control in the absence of Rb1. Further, compound There is evidence that the effects of pRb on cell loss of E2F1 or , important targets for pRb- proliferation and survival are separable in some mediated cell cycle control, can partially rescue both the biological contexts. For example, compound loss of unscheduled cell proliferation and excessive apoptosis Rb1 and E2F1 partially rescues the proliferative defects phenotypes (Tsai et al., 1998; Ziebold et al., 2001; in both the central and peripheral nervous systems, but Saavedra et al., 2002). Ectopic expression of E2F1 has only causes a significant reduction of apoptosis in the long been known to sensitize cells to apoptotic cell death central nervous system (Tsai et al., 1998). In contrast, (Shan et al., 1996; Agah et al., 1997; DeGregori et al., compound loss of E2F3 significantly diminishes apop- 1997), and cells lacking E2F1 are resistant to some tosis observed in the peripheral nervous system in the apoptotic triggers (Field et al., 1996; Leone et al., 2001). absence of Rb1 (Ziebold et al., 2001). Conditional abla- Ectopic expression of E2F3 can also induce apoptosis, tion of Rb1 in the nervous system elicits unscheduled

Oncogene Rb1 functions unrelated to the cell cycle DW Goodrich 5238 cell proliferation as expected, but does not cause free DNA ends. Topoisomerase II and pRb are capable elevated apoptosis (Ferguson et al., 2002; MacPherson of binding one another, and loss of pRb slows the et al., 2003). Hence cell proliferation and apoptosis are degradation of etoposide trapped TopII and the repair not necessarily coupled upon pRb loss. Another of the resulting DNA break (Xiao and Goodrich, 2005). hypothesis proposed to reconcile these results with the As a result, cells lacking pRb are more sensitive to the previously demonstrated involvement of pRb/E2F cytotoxic effects of TopII than cells expressing pRb. complexes is that apoptosis requires a cellular stress in Efficient degradation and repair of trapped TopII addition to pRb loss. Hypoxia caused by defective lesions correlates with BRCA1/TopII interaction, placental development and hematopoiesis may provide and BRCA1/TopII interaction requires the presence of the required cellular stress. The stress response gene p53 pRb. BRCA1 complexes not only have responds to hypoxia and is activated in the central activity, but also contain DNA repair factors. This nervous system of Rb1 nullizygous embryos. Compound suggests a model wherein pRb recruits factors like loss of p53 diminishes apoptosis in the central nervous BRCA1 complexes to trapped TopII lesions (Figure 2). system caused by Rb1 loss (Macleod et al., 1996). Hence These factors facilitate the degradation of trapped TopII any condition that diminishes cellular stress or the stress and the repair of the resulting DNA break. Whereas response is expected to reduce apoptosis in the absence pRb is also necessary for cell cycle checkpoint activation of pRb. in response to etoposide, processing and repair of Another piece of circumstantial evidence that sup- trapped TopII complexes is independent of cell cycle ports the hypothesis that pRb is more directly involved checkpoint activation. The E2F1–3 binding deficient in the regulation of apoptosis is that pRb is a substrate R654W pRb mutant is still capable of facilitating for caspases (Fattman et al., 1997, 2001; Tan et al., 1997; processing and repair of trapped TopII lesions despite Boutillier et al., 2000). Caspases are activated to its inability to trigger the normal cell cycle checkpoint transduce death signals and execute cell killing during response (Xiao and Goodrich, 2005). The above analysis apoptosis. Cleavage of pRb by caspases can either suggests pRb may use multiple mechanisms to directly inactivate the protein, or possibly activate novel pRb regulate cell survival in response to cellular stress, regulatory functions (Lemaire et al., 2005). Engineered including stress induced by cancer chemotherapeutics. mutations that block cleavage at particular caspase These mechanisms may be dependent or independent recognition sites within pRb have been shown to of E2F and the cell cycle. attenuate apoptosis in vitro induced by tumor factor-a (Tan et al., 1997) or potassium deprivation (Boutillier et al., 2000). Inhibition of apoptosis is The pRb-associated proteome unrelated to the cell cycle since the cell cycle response to tumor necrosis factor-a is the same in cells expressing At the molecular level, pRb function can be understood wild-type or the mutant pRb. The physiological through the proteins it interacts with and the functional relevance of pRb caspase cleavage has been demon- consequences of those interactions. Our current percep- strated by introduction of an analogous mutation into the mouse (Chau et al., 2002; Borges et al., 2005). The caspase cleavage resistant Rb1 allele supports normal growth and development, but mice containing this mutation exhibit tissue-specific resistance to apoptosis when challenged with bacterial endotoxin. The caspase resistant Rb1 allele also promotes colorectal cancer. It is of interest that the caspase cleavage resistant pRb does not inhibit apoptosis triggered by other apoptotic stresses like DNA damage. Rb1 has long been known to influence sensitivity to DNA damaging agents as pRb deficient cells have increased sensitivity to cisplatin, methotrexate, etopo- side, and camptothecin among others (Almasan et al., 1995; Harrington et al., 1998; Knudsen et al., 2000). In particular, pRb is required to efficiently trigger cell cycle checkpoints in response to DNA damage. In the absence of checkpoint activation, cells usually undergo apoptotic cell death. This function of pRb can be accounted for Figure 2 A model for a direct role for pRb in repair of trapped Topoisomerase II (TopII) DNA lesions. Rb1 protein is suggested to by the pRb/E2F mechanism, but other evidence has serve as an adaptor protein to recruit processing and repair factors suggested that more direct mechanisms may also be to trapped TOP2 cleavage complexes. BRCA1/BARD1 E3 involved. Topoisomerase II (TopII) poisons like etopo- ubiquitin ligase activity facilitates degradation of TOP2 to reveal side create a unique double-stranded DNA break where free DSBs that are then repaired by BRCA1-associated double- stranded DNA break repair factors. The model is consistent with the ends of the DNA are trapped in covalent linkage the observed requirement for an E2F- and cell cycle-independent with TopII. In order for this damage to be repaired, the pRb activity in the efficient processing and repair of etoposide- covalently linked TopII must be degraded to liberate induced DNA lesions.

Oncogene Rb1 functions unrelated to the cell cycle DW Goodrich 5239 tion of Rb1 function is based on an E2F-centric view of the literature that supports such a reshuffling of pocket the underlying molecular mechanisms. This is under- protein complexes upon experimental perturbation. For standable considering that the E2F transcription factors example, loss of E2F4, the preferred E2F binding were the first cellular pRb-associated proteins identified. partner for p107 and p130, results in the formation of Further, the functional consequences of the pRb/E2F novel p107/E2F1–3 and p130/E2F1–3 complexes that interactions are the most thoroughly elaborated. The are not detected under normal conditions. These novel biological importance of this mechanism, particularly complexes have functional consequences as they can for pRb-mediated cell cycle control, has consistently partially suppress tumorigenesis initiated by Rb1 loss been validated in numerous in vitro and in vivo studies (Lee et al., 2002). Further, specific disruption of pRb/ over the last decade. Yet, by one important criterion, E2F1–3 interactions by mutation can augment the this view of Rb1 is lacking. With the possible exception formation of other pRb complexes, presumably because of the retina, we are still unable to reasonably predict E2F1–3 no longer compete for pRb binding (Sun et al., what effect Rb1 pathway deregulation will have on 2005). It can be inferred from these observations that tumorigenesis in a given tissue in mice or in man. This disruption of particular pocket protein interactions will limits our ability to rationally target this important not only result in loss of function, but may also yield tumor-suppressor gene pathway for therapeutic benefit. gain of function as novel complexes form due to altered How best to realize a molecular understanding of pRb stoichiometric conditions within the cell. Viewing pocket sufficient to achieve this goal? There are still a protein function from the more global perspective of sufficiently large number of gaps in our understanding a highly interconnected protein interaction network may of pRb/E2F-dependent mechanisms and their regulation be required in order to accurately predict the con- to suggest that a continued focus on E2F may yield sequences of pRb inactivation in a particular biological the desired result. However, what of the potentially context (Figure 3). important E2F-indpendent mechanisms that are being identified? How many additional pRb-mediated me- chanisms are yet to be discovered? A previous survey of Perspectives the published literature has identified some 110 cellular proteins that have been implicated to physically interact Consideration of pRb function as a component of such with pRb (Morris and Dyson, 2001). Roughly 15% of a complex and interdependent protein interaction net- these proteins are kinases or phosphatases that could work has both theoretical and practical implications potentially regulate pRb activity, or the activity of pRb- for our understanding of cancer and its treatment. Many associated proteins. About two-thirds of the proteins pocket protein binding partners are restricted to are involved in transcription. The remaining 20% have particular tissues and cell types. Hence the distribution varied functions that are not obviously related to of distinct pocket protein complexes will vary from one transcription. In the ensuing 4 years since this study cell type to another. The biological consequence of pRb appeared, more than 40 additional cellular proteins have loss in a particular cell will be determined by the protein been proposed in the published literature to physically interactions that are lost, and how the liberation of these interact with pRb, with the grand total now exceeding pRb-associated proteins affects the distribution of the 150 different cellular proteins. This represents an remaining pocket protein complexes. In other words, average discovery rate of more than 10 new pRb- pRb may suppress tumorigenesis in different ways that associated proteins per year. At least eleven new pRb- vary depending on the tissue and cell type. The net effect associated proteins have been newly described in 2005, of pRb loss in a given cell will also be determined by the so the rate of pRb-associated protein discovery is not status of independent regulatory pathways. For exam- diminishing. It is unclear how many of these pRb ple, it has been proposed that the cell of origin for protein interactions form the basis for biologically retinoblastoma is naturally resistant to apoptosis (Chen relevant molecular mechanisms. Yet, even by conserva- et al., 2004; Zhang et al., 2004b). This may explain why tive estimate, dozens of distinct pRb protein complexes complete pRb loss in this cell initiates retinoblastoma may be operable at any one time in a given cell. This rather than cell death. A practical consequence of this is is plausible given that pRb is generally in significant that pRb inactivation may have different prognostic stoichiometric excess over most if its protein binding implications for different cancers. This could occur, for partners. example, if pRb inactivation has a different effect on the Another consideration is the existence of the structu- response to cancer therapy in one tissue than it does in rally and functionally related pocket proteins p107 and another. p130. The cellular proteins that interact with p107 or How the Rb1 pathway or network is deregulated p130 are largely undefined, but it is clear that they can during carcinogenesis, either direct mutation of Rb1 or bind some of the same proteins that associate with pRb. mutation of genes that regulate pRb, is not randomly Hence the pocket proteins may be in competition for the distributed in human cancer (Figure 4). Retinoblastoma, binding of an overlapping subset of cellular proteins. small-cell , and osteosarcoma are commonly Given this, any perturbation of the pocket proteins will associated with Rb1 mutation. In contrast, Rb1 muta- have a substantial ripple effect based on a reshuffling of tion is rare in . Melanoma is commonly asso- pocket protein complexes due to the altered stoichio- ciated with perturbations of or p16Ink4a that metry of unbound protein partners. Evidence exists in lead to unscheduled pocket protein phosphorylation.

Oncogene Rb1 functions unrelated to the cell cycle DW Goodrich 5240

Figure 4 Mechanisms of pRb inactivation in human cancer. Three main mechanisms of pRb inactivation are observed in human cancer, and these mechanisms are not randomly distributed. Rb1 mutation is common in some cancers, but rare in others. Likewise, inactivation by deregulated pRb phosphorylation is common in a Figure 3 The pocket protein interaction network. While the pRb/ distinct subset of human cancers, but Rb1 mutation is rarely E2F complex is central to pRb-mediated cell cycle control and observed in this subset. The model proposes that each mechanism contributes to pRb-dependent differentiation and cell survival, of inactivation has different molecular and functional conse- more than 150 additional pRb protein binding partners have been quences. Deregulated pocket protein phosphorylation is proposed described in the literature. The other pocket proteins, p107 and to abrogate some protein interactions but not others. Coupled with p130, interact with an overlapping subset of these cellular proteins. considerations of the pocket protein interaction network outlined The model pictured attempts to illustrate some of the functional in Figure 3, different cell types may select for different mechanisms implications of this interdependent protein interaction network. of pRb inactivation because the molecular consequences are more Cell A and cell B represent two different cell types that express favorable for carcinogenesis. different constellations pocket protein complexes. Hence the function of pRb in these two cells is different. In cell A, p107 and p130 are either absent or they are unable to interact with the deregulation of the kinases that regulate multiple pocket pRb-associated proteins shown. In this simple case, Rb1 loss results proteins would be selected for. Hence the consequences in the loss of all functions mediated by the indicated protein of pRb incativation will not only be dependent on cell interactions. Cell B expresses a unique set of cellular proteins that interact with each of the pocket proteins with differing affinities. type, but also on the mechanism of inactivation. Thus, Based on these differing affinities and simple stoichiometric the behavior of cancers of a given type may vary considerations, loss of Rb1 causes a reshuffling of pocket protein depending on the nature the perturbation in the pRb complexes resulting in loss of some functions (W, Z), gain of other network. For example, with complete functions (Y), or no significant change in function (E2F-functional compensation). Even more complicated scenarios can be envi- loss of pRb behave differently than partially penetrant sioned upon partial inactivation of pocket proteins by phosphory- cases that express mutant pRb or low levels of wild-type lation. pRb. This concept may apply more broadly to the common adult cancers suggesting that subclassification by the nature of the lesion in the pocket protein This clustering suggests that different types of interaction network may be informative. cancers select for different mechanisms of pRb inactiva- The simple genetics of retinoblastoma is a rare tion. The implication is that the different mechanisms of exception in human cancer. Most common adult cancers pRb inactivation yield different functional conse- arise through alterations in multiple combinations of quences. A relevant example may be the ability of pRb many genes. The molecular cloning of the Rb1 gene has to negatively regulate apoptotic cell death. As discussed been met with great fanfare since it provides, in a sense, above, the cell of origin for retinoblastoma may be a simple model system in which to study the molecular naturally resistant to apoptotic cell death. - basis for neoplastic transformation. Mutational inacti- esis in such a cell may benefit from complete loss of pRb vation of a single gene is apparently necessary and rate since the potential proapoptotic consequences are irrele- limiting for the genesis of a human cancer. In the vant. In contrast, the cell of origin for melanoma may ensuing 20 years, significant progress has been made possess robust apoptotic responses. Carcinogenesis in in the characterization of pRb as a general cell such a cell would select against complete loss of pRb since cycle regulator, elucidating the molecular mechanisms the ensuing apoptotic response would preclude tumor responsible for this regulation, and accounting for progression. In this case, deregulated phosphorylation how this activity may be regulated by phosphorylation. of pRb may partially inactivate critical tumor-suppres- Our current level of understanding, however, does sion functions, but not the antiapoptotic functions. not explain a number of nagging questions. Why is Alternatively, other pocket proteins may also need to Rb1 a tissue-specific tumor suppressor given its more be partially or completely inactivated. In this case, general role in cell cycle regulation? Why among the

Oncogene Rb1 functions unrelated to the cell cycle DW Goodrich 5241 functionally related pocket proteins is mutation of Rb1 typical of most human cancer. In a sense, Rb1 is the uniquely associated with human cancer? Why does the exception that proves the rule that a large number of mechanism of pRb inactivation cluster in particular interdependent molecular alterations are required for types of cancer? If pRb is fundamentally a cell cycle neoplastic transformation. Yet, pRb remains a con- regulator, why does it bind so many cellular proteins venient starting point for addressing this complexity. with seemingly divergent functions? What do these other Applying the experimental and computational ap- protein interactions contribute to pRb-mediated tumor proaches being rapidly developed in the field of protein suppression? A more complete consideration of cell interactome mapping (Vidal, 2005) to the pocket protein cycle independent pRb functions may be necessary to network may yet helpus justify the scientific attention answer these questions. paid this important tumor-suppressor gene over the two Ultimately, it seems, pRb function is considerably decades since its discovery. more complex than is currently understood. Rb1 is seemingly involved in many fundamentally important cellular processes including the cycle, Acknowledgements cellular stress responses, differentiation, cellular senes- cence, and programmed cell death, among others. The I am grateful to the Goodrich lab members and colleagues Rb1-encoded protein utilizes a large and complex Roswell Park Cancer Institute for stimulating discussions. array of protein interactions to influence these processes. This work was supported by a grant from the National Cancer This complexity is reminiscent of the genetic complexity Institute (CA70292).

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