Retinoblastoma, Molecular Genetics ofUpdated by:Timothy W. Corson1 and Helen Dimaras2 ,3,4 1 Indiana University School of Medicine, Indianapolis, USA2 University of Toronto, Toronto, Canada3 Toronto Western Research Institute, Toronto, Canada4 The Hospital for Sick Children, Toronto, Canada

Vivette D. Brown1 and Brenda L. Gallie1,2,31Princess Margaret Hospital, Toronto, Canada2Hospital for Sick Children, Toronto, Canada3University of Toronto, Toronto, Canada

1 Retinoblastoma, Cancer of Infants 3492 Genetic Principles of Cancer Discovered through Retinoblastoma 3502.1 Genetic Locus of the Retinoblastoma Gene 3502.2 Retinoma 3513 Spectrum of RB1 Mutant Alleles and Risk Prediction 3514 The Retinoblastoma Gene and Protein 3524.1 RB ProteinpRB Structure 3534.2 RB Family Members 3544.3 RB ProteinpRB Phosphorylation 3554.4 RB and E2F Protein Families in Cell Cycle Progression 3574.5 Other Binding Proteins 3584.6 Differentiation by Retinoblastoma Protein 3595 RB Family Regulation of Murine Development 3605.1 RB1 in Murine Development 3605.2 p107 and p130 in Murine Development 3605.3 E2F in Murine Development 3626 Retinoblastoma in Mice 3626.1 Viral Oncoprotein-induced Retinoblastoma 3626.2 Chimeric Knockout Retinoblastoma 3636.3 Targeted RB1−/− Knockout Retinoblastoma 3637 RB ProteinpRB and Apoptosis 3648 Additional Genomic Changes in Retinoblastoma 3658.1 Genomic Gains and Losses 3658.2 Candidate M3-Mn Genes 3668.3 Causes of Drug Resistance in Retinoblastoma 3669 Importance of Retinoblastoma 367

Bibliography 368Books and Reviews 368Primary Literature 368

KeywordsApoptosisThe process of programmed cell death.E2FA transcription factor that regulates S-phase genes.LOHLoss of heterozygosity.MYCNAn oncogene…ProbandThe very first individual in a family to be diagnosed.RB1Retinoblastoma tumor susceptibility gene.Retinoblast

A precursor cell that becomes a retinal cell.SV40Simian virus 40 DNA tumor virus.TAgSimian virus 40 large T antigen oncoprotein.Tumor SuppressorA gene that prevents protects a cell from becoming a tumor.

Retinoblastoma is a rare malignant tumor, primarily initiated by the inactivation of both alleles of the retinoblastoma tumor susceptibility gene, RB1, in a developing human retinal cell. Surprisingly, the protein (pRB) encoded by RB1 is an important transcription factor. Cell cycle control by pRB is mainly accomplished by transcriptional repression of genes required for cell cycle progression. Control of differentiation by pRB is achieved by the activation of transcription. Almost every kind of tumor has disruption in the retinoblastoma pathway associated with tumor progression, but germline mutation of the RB1 gene predisposes children to a 95% specific risk of developing retinoblastoma and a significant but much less ly increased risk of second primary tumors, such as osteosarcoma and melanoma. A less common subset of retinoblastoma is initiated by somatic amplification of the MYCN oncogene in a predisposing retinal cell. Individuals affected by MYCN-amplified retinoblastoma face non of the retinoblastoma associated hereditary and second-cancer risks. Ongoing intense efforts to define the features of the retinal cell that is sensitive to RB1 loss have potential to once again broadly influence the understanding of cancer in general.

1 Retinoblastoma, Cancer of InfantsRetinoblastoma most commonly is diagnosed only when tumor fills one or both eyes, and the parents notice a white gleam from the pupil of the baby’s eye. Often, the diagnosis is definitive based on clinical examination and imagingCT Scan that reveals calcification within the tumor. When only one eye is affected, the preferred treatment is almost always removal of the eye, which is usually curative. When both eyes are involved, the choice of treatment for each eye depends on the size and extent of tumor, the risk of tumor spreading outside the eye, and the risk to vision of the treatment. The new International Intraocular Retinoblastoma Classification (IIRC), which stages each eye with retinoblastoma and predicts the likelihood of treatment curing the eye, is being validated through a global Internet survey.

Eyes with advanced tumor at risk to spread outside the eye, and virtually no possibility of useful vision even if the tumor is controlled (IIRC Group E), are removed. Eyes with medium or large tumors (IIRC Groups B, C, D), no risk of extra-ocular tumor and some possibility of vision, are now commonly treated by systemic or local (peri-ocular, intravitreal, or intraarterial) chemotherapy combined with laser and cryotherapy to destroy physically

any surviving or chemotherapy-resistant tumor cell. Eyes with small tumors not immediately adjacent to the center of vision or the optic nerve (IIRC Group A) are treated with laser and cryotherapy. In recent years, chemotherapy has also been delivered directly to the eye by intravitreal, intraarterial, and periocular routes. Cure is usually obtained within 2 to 4 years. Late recurrences are rare. The Where resources and expertise are available, the fraction of children who lose both eyes to retinoblastoma is now small, and m. Most children end up with vision in at least one eye. However globally this is not the case; most children with retinoblastoma live in resource-limited countries, where chance of survival, let alone vision-salvage, is low.

Retinoblastoma tumor cells may escape from the eye and spread through the optic nerve to the brain or through the blood to grow preferentially in the bone marrow. Accurate histology of enucleated eyes is essential to determine if tumor has invaded the structures of the eye and to what extent. [Refernece pTNM staging, role in prescribing adjuvant chemotherapy and survellaillance]. Surveillance of bone marrow and CSF… Until recently, metastatic retinoblastoma was incurable. There are now several reports of long remissions and, possibly, cure following intensive systemic chemotherapy, chemotherapy given into the meningeal space, and megadoses of chemotherapy to destroy any surviving tumor cells followed by stem cell transplant to reconstitute the bone marrow.

Radiation therapy is very effective in controlling retinoblastoma tumor growth. However, radiation of children with RB1 mutant alleles greatly increases the risk of a second primary cancer later in life, from the already high risk that the mutant RB1 allele imposes. Some agents used in Chemotherapy chemotherapy for retinoblastoma also carryies a risk of induction of leukemia, but the magnitude of this risk has not been established in the context of therapy for retinoblastoma.

2 Genetic Principles of Cancer Discovered through RetinoblastomaThe tendency for retinoblastoma to be transmitted in families as an autosomal dominant trait was recognized in the early 1900s. The location of the predisposing gene on chromosome 13q was suggested by the observation in the early 1960s that large deletions involving chromosome 13q14 were associated with retinoblastoma

and other abnormalities. However, bilateral retinoblastoma was thought to be a unique special case of cancer in which genetics played some role, while non-genetic mechanisms were presumed, erroneously, to underlie common forms of cancer.

In 1971, Alfred Knudson proposed, from simple analysis of age of diagnosis of bilateral versus unilateral retinoblastoma, that both hereditary (usually bilateral) and nonhereditary (always unilateral) retinoblastoma are initiated by two mutational events, M1 and M2. In 1973, Comings proposed the possibility that these two events could be the two alleles of one gene whose normal function would be suppression of tumors.

2.1 Genetic Locus of the Retinoblastoma GeneProof that retinoblastoma tumors had mutation of both alleles of the 13q14 predisposing gene was obtained when genetic markers could were developed to distinguish the two alleles. Although the RB1 gene was not yet cloned, markers near the RB chromosomal locus showed that the heterozygous 13q14 allele associated with the presence of retinoblastoma in members of a family was often the only retained allele in retinoblastoma tumors (loss of heterozygosity, LOH). LOH for 13q14 was observed in both hereditary and unilateral, likely not hereditary, retinoblastoma tumors, proving that the Coming’s predictions are of Comings correct. Both hereditary and nonhereditary retinoblastomas are nearly always initiated by mutation of the same tumor suppressor gene, the RB1 gene (see below for an exception to this rule). The retinoblastoma studies opened the way to find other tumor suppressor genes in which mutation initiates a wide variety of other cancers. Through the 1980s, numerous papers reported the chromosomal localization of disease-specific tumor suppressor genes by searching for LOH in tumors. Cancer was clearly demonstrated to be the most common somatic genetic disease.

In sporadic bilateral retinoblastoma patients, the M1 occurs either de novo in a parent and is transmitted via the germ cells or during embryonal development. Retinoblastoma is initiated when the remaining normal allele is inactivated (M2) in a retinoblast developing retinal cell carrying M1. Sporadic unilateral retinoblastoma occurs in children with normal RB1 germline alleles, who develop rare somatic mutations of both alleles (M1/M2) in a developing retinal cell.

Bilaterally affected patients all have one germline RB1 mutant allele, which results in autosomal dominant transmission of the predisposition to retinoblastoma. Unilaterally affected patients have only a 15% chance that they carry a germline RB1 mutant allele. Therefore, the standard approach for unilateral patients is to test the retinoblastoma tumor DNA to identify the two mutant RB1 alleles and then test the blood DNA for each identified mutant allele. In this way, a negative result generally rules out heritable retinoblastoma in the unilateral proband, although mosaicism is never excluded. About 10% of new RB1 germline mutations can be shown to be mosaic, present in less than 50% of the RB1 DNA and in only a fraction of the cells of the proband. Clinically, mosaicism may become obvious when a parent has more than one child with retinoblastoma but does not show in blood the mutant RB1 allele present in the children. Mosaicism cannot be inherited since the predisposing mutation must occur during the development of the embryo. Antecedent By DNA analysis, antecedent relatives are excluded from risk, and future offspring of the unilateral patient can be tested for the mutant alleles found in the tumor of their parent, just in case the parent had undetected germline mosaicism.

The majority of children with heritable retinoblastoma have newly acquired RB1 mutations, and thus no family history of the disease. Most frequently, it is the paternal RB1 allele that is mutated, presumably because of the increased risk of mutation of sperm relative to ova. About 10% of new RB1 germline mutations can be shown to bemosaic, present in less than 50% of the RB1 DNA and in only a fraction of the cells of the proband. Clinically, mosaicism may become obvious when a parent has more than one child with retinoblastoma but does not show in blood the mutant RB1 allele present in the children. Mosaicism cannot be inherited since the predisposing mutation must occur during the development of the embryo.

2.2 RetinomaNonprogressing retinal lesions were originally considered to represent ‘‘“spontaneous regression’’ regression” of retinoblastoma. Retinoma is a preferable name, which does not imply knowledge of a mechanism. Also known as retinoma (sometimes called retinocytoma) this lesion clinically presents asis typically a translucent gray retinal mass, frequently associated with calcification and hyperplasia of the retinal pigment epithelium, unlike the actively proliferating retinoblastoma that shows calcification but appears opaque white. Pathology On histopathology,

of retinoma (also called retinocytoma) shows a varied arrangement of non-proliferative neuronal cells with photoreceptor differentiation in the form of fleurettes. Unlike regressed tumors, retinoma does not show evidence of necrosis, axons, Müller glia cells, and astrocytes. [Add in something about RB pathology here?]In contrast, retinoblastoma is highly proliferative, its cells crowding together in a cobblestone pattern, and sometimes shows differentiation in the form of Flexner-Wintersteiner or Homer Wright rosettes. Retinoblastoma also often shows pseudorosettes, which are radial arrangements of cells around blood vessels; cells too far away from the blood supply tend to necrose.

Although retinomas are ‘‘“benign,’’ ” they can undergo malignant progression to retinoblastoma. Particularly in a young child, such tumors must be watched regularly for indication of transition to retinoblastoma. RB1 germline mutations predispose to both retinoma and retinoblastoma in the same patient and in other members of a family who have the mutant RB1 allele. Careful molecular analysis showed that retinomas do indeed have mutation of two RB1 alleles, but lack some of the subsequent genetic changes seen in retinoblastoma (see below), strongly suggesting that they are a “premalignant” lesion rather than spontaneous regression.Although nomolecular analysis of retinoma has been reported, germline RB1 mutations have been identified in several patients with bilateral or unilateral multifocal retinomas as their only clinical manifestation.

2.3 MYCN-amplified retinoblastomaApproximately 1% of unilateral tumors are not initiated by mutation in RB1, but by amplification of the MYCN oncogene….

3 Spectrum of RB1 Mutant Alleles and Risk PredictionRB1 mutations are spread throughout the gene including the promoter, most exons, and splicing regions of introns (Fig. 1). More than 50% of RB1 mutations have been found only once, and the majority of germline mutations originate in the proband. Molecular identification of RB1 mutations supports accurate determination of the risk of retinoblastoma in relatives.

RB1 mutations are most frequently ‘‘null’’ mutations that are out of frame and result in premature STOP codons (Fig. 1). Premature truncation of translation in turn triggers nonsense-mediated decay, unstable mutant mRNA, and no detectable pRB protein. The only significant hot spots for RB1 mutations are the ‘‘recurrent’’ mutations,

Fig. 1 Retinoblastoma-initiating mutations are distributed throughout the RB1 gene. Of the 230 germline mutations, 53% have been reported only once. Recurrent mutations are shown in red, with the number of occurrences indicated in the box. In-frame mutations are shown in blue. Boxes indicate splice mutations. Circles indicate small point or few nucleotide mutations. Lines indicate lengths of deletions. (See color plate p. xxxiii).

most commonly CGA (arginine) mutating to TGA (STOP) because CpG codons are prone to mutate to TG. Missense mutations almost invariably involve the A/B ‘‘“pocket’’ pocket” domain of pRB, which is critical in many protein interactions of pRB.

In bilateral probands, 93% of the germline mutations are null, 6% are inframe, and 1% are promoter. In the uncommon unilateral germline probands (both familial and sporadic), the ratios of null (57%), in-frame (40%), and promoter (3%) mutations are very different. The presence of uA unilateral tumor in a patient with germline RB1 mutation indicates reduced expressivity of the germline RB1 mutation, manifesting as fewer than expected tumors, reducing the likelihood of a null allele. Reduced penetrance/expressivity RB1 mutant alleles retain enough activity to prevent some, but not all, retinoblastoma tumors. The types of mutant alleles identified in retinoblastoma tumors, presumably of somatic origin (88% null, 3% in-frame, and 8% promoter methylation), are different again.

The second RB1 allele (M2) is lost by LOH in 52% of sporadic retinoblastoma tumors. The likelihood that the M2 event is LOH also varies with the type of M1 event. While 50% of the tumors with M1 whole gene and exonic deletions show LOH, 88% of tumors with methylation of the promoter show LOH. The biological reasons for these differences are unknown.

4 The Retinoblastoma Gene and Protein

The RB1 gene was cloned by identification of a chromosome 13 DNA clone that was totally deleted from a retinoblastoma tumor. This was correctly interpreted to be LOH for a deleted allele, and the clone turned out to be located within an exon of the RB1 gene. The 190-kb RB1 gene transcribes a 4.7-kb RNA in 27 exons.Comparison of the human and mouse RB1 genes shows 80% homology in the first 235-bp upstream of the initiating methionine, 89% homology in the coding sequence, and 77% homology in the 3’ untranslated region. The human and mouse promoters contain binding sites for the transcriptional elements ATF, SP1, and E2F, but are devoid of TATA and CAAT boxes. The human and mouse proteins show 91% identity, suggesting that the function of this protein is highly conserved. The retinoblastoma protein (pRB) is a nuclear phosphoprotein of 928 amino acids that runs as a doublet of molecular weight 110–114 kDa on SDS polyacrylamide gel electrophoresis. The slower migrating band corresponds to the phosphorylated species of pRB.

RB1 function is abrogated either by deletion or mutation in retinoblastoma. In most other tumor types, RB1 is mutated or functionally inactivated, confirming that RB1 is a tumor suppressor gene. The reintroduction of RB1 into certain cells that have nonfunctional RB1 alleles can suppress proliferation. For example, retroviral introduction of RB1 into the retinoblastoma cell line WERI-Rb27, the osteosarcoma cell line Saos2, the prostate carcinoma cell line DU145, and the bladder carcinoma cell line HTB9 resulted in morphological changes, reduction in growth rate, reduced colony formation in soft agar, and reduced tumorigenicity in nude mice. However, introduction of pRB into the U2OS osteosarcoma cell line, which contains a normal retinoblastoma allele, or the C33A cervical carcinoma cell line that contains a mutant pRB, had no effect. The ability of pRB to reverse the malignant phenotype of tumor cells may depend on what other genetic alterations have occurred in the cell.

4.1 RB ProteinpRB StructureFunctional domains of the coding region of pRB were delineated using deletion mutant analysis. The protein is divided into four regions, the N terminus, the A and B domains separated by a spacer region, and the C terminus (Fig. 2). Disruption of growth suppression by pRB requires the A and B domains as well as portions of the C-terminal domain. The nuclear c-Abl protein binds to the C terminus of pRB, promoting proliferation. The C terminus is required for growth suppression along with the A and B domains.

The A/B region of pRB binds to the LXCXE (Leucine-X-Cysteine-X-Glutamic acid, where X is any amino acid) epitope of the viral oncoproteins, simian virus 40 large T antigen (TAg), adenovirus E1A, and human papilloma virus-16 E7. The importance of the A/B domain is evident by the large number of endogenous proteins binding to it and the number of tumorigenic missense mutations affecting it (Fig. 1). X-ray crystallography shows that the A domain is required for stable folding of the B region, the site of binding of LXCXE proteins. Even when the A and B regions of pRB are expressed on separate proteins, they are able to form a repressor motif that is regulated by phosphorylation. Another highly conserved region is the intervening sequence between the A and B regions, suggesting an additional protein binding site, or a critical role in folding. Indeed, the E2F transcription factor (see below) binds to the highly conserved interface between the A and B regions of pRB.Fig. 2 Structure of pRB. Numbers above the schematic designate the amino acids. Mu corresponds to murine pRB (normal numbers) and hu to human pRB (italics). The N and C termini and the A and B regions are required for binding the SV40 large T antigen. Positions of the 16 phosphorylation sites in murine pRB are shown with S (serine) (red) and T (threonine) (blue). Areas of pRB required for different functions are indicated below the schematic. (See color plate p. xxxiii).

The ability of pRB to enter the nucleus is regulated by a bipartite nuclear localization signal located in the C terminus. The underphosphorylated (active) form of pRB binds the nucleus, following salt extraction, more effectively than the phosphorylated (inactive) form. A/B region mutants are poorly retained in the nucleus, and the A/B region can experimentally translocate a cytoplasmic protein to the nucleus.

4.2 RB Family MembersTwo genes/proteins homologous to RB1pRB, p107 (encoded by RBL1), and p130 (encoded by RBL2), which had previously been observed to bind E1A, were identified by sequence comparison. Some antibodies that were developed against pRB also recognized

p107 and p130, suggesting structural homology. All three RB family proteins are found in both hypo- and hyperphosphorylated forms. However, while p107 and p130 bind to cyclin A and cyclin E, pRB does not. Cyclin A binds to the A/B spacer region of p107, which is highly conserved between p107 and p130.

The RB family members express differently during the cell cycle. During quiescence, p130 is expressed, but disappears as cells are stimulated to reenter the cell cycle. Quiescent cells do not express p107, but as cells reenter the cell cycle, p107 accumulates. In contrast, pRB levels are generally constant throughout the cell cycle, although there is a small increase in the level of pRB following growth stimulation.

While tumor suppression was defined first by the powerful function of pRB, there is not much evidence that the other RB family members can independently act as tumor suppressors. However, to some extent, the RB family compensate for each other. Exogenous overexpression of p107 can suppress the growth of C33A cells, which have a mutant pRB, even though wild-type pRB is unable to. Growth suppression in C33A cells by p107 is mediated through its cyclin/cdk binding domain in addition to the A/B and C domains. p130 has been shown to suppress the growth of only one cell line, the human glioblastoma cell line T98G, which is refractory to suppression by pRB or p107. Few mutations of p107 or p130 have been observed in human tumors.

4.3 RB ProteinpRB PhosphorylationOne of the first clues to the function of pRB came following the discovery that the phosphorylation status of pRB depends on the cell cycle. pRB is underphosphorylated in G0/G1 phase and hyperphosphorylated in the S- and G2/M phases in all cell types. pRB phosphorylation occurs on serine and threonine residues, with up to 16 potential sites. No phosphotyrosine is evident. Following mitogenic stimulation, pRB is phosphorylated in three stages during cell cycle entry and progression: mid-G1-phase, S-phase, andG2/M (Fig. 3). Dephosphorylation of pRB occurs after the beginning of mitosis, and may be regulated by differentiation signals.

Because of its significance in growth regulation, great attention has been paid to the regulation of pRB by phosphorylation. Large T antigen specifically binds unphosphorylated (active) pRB to transform cells, possibly by releasing cellular-binding proteins. Inactivation of pRB by phosphorylation occurs through the action of the cyclin-dependent kinases (cdks). The cdk family member, p34cdc2, was first found to bind to and phosphorylate pRB. The cdc2 kinase is the active subunit of the M-phase promoting factor, which phosphorylates specific serines or threonines in its consensus site Z-S/T-P-X-Z (where X is polar and Z is generally basic). pRB contains 16 potential cdc2 phosphorylation sites, of which at least nine have been shown to be phosphorylated in vivo (Fig. 2). Phosphopeptide analysis shows that in vitro phosphorylation by cdc2 corresponds to in vivo phosphorylation of pRB. However, cdc2 is activated in late G2-and M-phases and a homologous kinase, cdk2, may first bind to and phosphorylate pRB, consistent with phosphorylation of pRB to promote exit from G1. Despite multiple phosphorylation sites potentially offering subtle regulation of pRB transcriptional inhibition and protein-protein interactions, crystal structures have indicated that T373 and S608 are particularly crucial residues. Phosphorylation of T373 stimulates a global conformational change, while S608 phosphorylation orders a flexible loop to block binding to the E2F transactivation domain.

Cdks are activated following binding to cyclins and phosphorylation of a conserved threonine by the Cdk-activating kinase. The expression of cyclins varies during the cell cycle, with cyclin D and E expressedduring G1, cyclin A during S-phase, and cyclin B during mitosis. Cyclins E and A form a functional unit by binding to cdk2 in G1- or S-phase respectively, whereas cyclin D forms a complex with cdk4 or cdk6 in G1. Cyclin B binds to cdc2 in late G2.

While complexes of each of these cyclins can phosphorylate pRB, only cyclins A, D, and E are able to reverse the ability of pRB to block cells in G1. Although initial reports suggested that phosphorylation of pRB occurred on the same residues whether phosphorylated by cyclin D-cdk4, cyclin E-cdk2, or cyclin A-cdk2 complexes, it is now recognized that different cyclin-cdk complexes regulate different phosphorylation sites, in a collaborative effort to completely inactivate pRB. In yeast, three G1 cyclins, Cln1, Cln2, and Cln3, regulate the activity of Cdc28 throughout G1. Mammalian cyclin E is able to substitute for a cln2 mutant and cyclin D1 for a cln3 mutant. Phosphorylation of pRB in yeast requires a combination of Cln3 and either Cln1 or Cln2, suggesting that cyclins D and E must collaborate to completely phosphorylate pRB. Study of cyclin D-cdk4/6 and cyclin E-cdk2 kinase inhibitors activities in mammalian cells confirms these results. In U2OS cells, pRB is partially phosphorylated by cyclin D kinase activity, with complete

phosphorylation requiring cyclin E activity. Phosphorylation of pRB only by cyclin D kinase did not release pRB from its interaction with E2F1. Continued phosphorylation of pRB on specific sites in S-phase by cyclin A-cdk2 activity may also be important for the completion of DNA synthesis.

pRB is also regulated by another kinase, cyclin C-cdk3, at the G0/G1 transition. Phosphorylation of pRB at specific substrate sites is required for cells to exit G0 efficiently (Fig. 3). Cyclin C directs phosphorylation of pRB in a temporal pattern that precedes pRB phosphorylation by cyclin D or cyclin E complexes. Therefore, cyclin C plays an important role in regulating the G0-to-G1 transition in a

pRB-dependent manner. Fig. 3 pRB regulates cell cycle progression, differentiation, and apoptosis. Hypophosphorylated pRB represses genes necessary for S-phase entrance by repressing transcriptional activation by E2F, causing cell cycle arrest, and activates genes necessary for differentiation by binding cell type–specific transcription factors. Phosphorylation of pRB by cyclin C-cdk3 kinase stimulates exit from G0, and in late G1 phosphorylation by cyclin D-cdk4/6 and cyclin E-cdk2 kinases releases E2F, allowing cell cycle progression. Continual phosphorylation of pRB occurs by cyclin A-cdk2 and cyclin B-cdc2 complexes in S and G2 phases. pRB is activated in late mitosis by dephosphorylation. Inappropriate entry into S-phase in the absence of pRB induces apoptosis.

Dephosphorylation (reactivation) of pRB is dependent on the activity of phosphoprotein phosphatase type 1 (PP1), which associates with pRB in vivo during mitosis. Microinjection of PP1 into either the nucleus or the cytoplasm of cells synchronized in G1 increases the resistance of pRB to extraction from the nucleus and

correlates with inhibition of S-phase progression.

4.4 RB and E2F Protein Families in Cell Cycle ProgressionViral oncoproteins induce proliferation of mammalian cells by interfering with binding of pRB to cellular proteins. The first such cellular protein to be identified was the transcription factor E2F. The E2F transcription factor regulates the activity of promoters containing an E2F binding site, originally identified as the sequence element in the adenovirus E2 promoter that allowed transcription in mammalian cells. The E2F binding site (TTTSSCGC where S is C or G) has been identified in promoters of genes encoding S-phase-regulatory proteins such as DNA polymerase α, thymidylate synthase (TS), proliferating cell nuclear antigen (PCNA) and ribonucleotide reductase (RR), and cell cycle progression genes such as cyclin A, cyclin E, cdc2, and B-myb. The E2F1 and RB1 genes are also regulated by E2F activity, suggesting the existence of an autoregulatory loop in the RB/E2F pathway. pRB mutants that bind free E2F but do not repress transcription are unable to block S-phase entry. Co-expression of E2F1 with pRB releases the G1 block and pushes cells into S-phase. Thus, the primary way that pRB blocks the cell cycle in the G1-phase is through repression of E2F-responsive genes.

The E2F family of proteins consists of seven eight members (E2F1, -2, -3, -4, -5, -6, and -7, and -8). E2F1-5 proteins each contain a DNA binding domain, a transactivation domain, and a C terminus region that binds pRBor other pRB family members. The ability of E2F1-6 proteins to regulate transcription is also dependent on binding to the DP family of proteins. Two Three mammalian DP proteins (DP1, -2, and -4) bind E2F to enhance interaction with E2F binding sites and activate E2F responsive promoters. However, not all genes with an E2F site are activated by E2F. In vivo, E2F is released from the E2F binding site of the B-myb promoter at the same time that B-myb transcription is activated, suggesting that the E2F complex suppresses this promoter. E2F6 contains neither the transactivation domain nor the C terminus pRB binding region, but represses transcription by complexing with DP proteins to either actively repress or replace other E2F/DP complexes. E2F7 and E2F8 haves two DNA binding domains but binds the E2F binding consensus site independently of DP proteins. It They too does not have lack a transactivation domain or and the pRB binding region, but functions as a repressors of certain E2F-regulated genes. A single gene may be regulated by both activating and repressive E2Fs. Overexpression of E2F1 pushes cells into untimely S-phase, followed immediately by induction of apoptosis. pRB protects cells from apoptosis by complexing with E2F1. However, while a transactivation defective E2F1 mutant induced apoptosis, a DNA-binding defective E2F1 mutant did not, suggesting that the pRB/E2F1

complex may repress genes necessary for induction of apoptosis.Different RB family proteins bind E2F family proteins at different phases of the cell cycle.

pRB binds E2F1- 4, whereas p107 and p130 bind only E2F4 and 5. E2F4 or 5 are predominantly bound to p130 in quiescent G0 cells and to p107 in proliferating cells. pRB binds and regulates E2F1, 2, and 3 in G1 but binds to E2F4 when cells are induced to reenter the cell cycle.

The activity of E2F is also regulated in a cell cycle–dependent manner by degradation of free E2F protein. E2F1 and 4 have C-terminal sequences that target them for degradation via the ubiquitin proteasome pathway. Binding of pRB to E2F1 or p107 to E2F4 increases the half-life of the E2F protein. Endogenous E2F1

is more stable in G0/G1 when bound to pRB than in S-phase. This suggests that the RB family may maintain cells in G1 by assuring that E2F is present to repress promoters. E2F activity is also stabilized by acetylation. pRB also blocks recruitment of the pre-initiation complex to the promoter by E2F.

4.5 Other Binding ProteinsA diversity of action is evident from the large number of proteins that bind to pRB with different functions (Table 1). In addition to E2F, pRB binds a number of general transcription factors (ATF-2 and SP1) and cell type–specific transcription factors (Elf-1, PU-1, MyoD, and NFIL6). Generally, the binding of pRB represses transcriptional activity; however, binding to factors such as c-Jun, MyoD, C/EBP, and NF-IL6 has a positive effect on differentiation of specific cell types,Tab. 1 Some of the proteins that bind pRB.Function pRB-binding ProteinsCdk inhibitor p21Chromatin modulator BRG1Chromosome scaffold H-NucCorepressor CtIP, RbAp48, RBP1, HBP1Cyclins Cyclins CCND1, 2, 3, CCNA1, CCNB1, CCNCHistone deacetylase HDAC1, 2, 3Methyltransferase DNMT1Molecular chaperone hsp75Nuclear matrix Lamin A, p84Phosphatase PP-1a2Pol I transcription factor UBFPol II transcription factor TAFII250Pol III transcription factor TFIIIBPotential tumor suppressor Prohibitin, BRCA1Protein kinase RbKRegulator of p53 stability MDM2, MDM4Replication licensing factor MCM7Ser/Thr kinase cdc2, cdk2Signaling molecule Raf-1Transcription factor ATF-2, c-Jun, c-Myc, N-Myc, C/EBP, E2F-1-3, Elf-1,

hBRM, Id-2, MyoD, Myogenin, NF-IL6, PU.1, Sp1, Pax-3, PHox, Chx10, RIZ

Transformation inducer BogTyrosine kinase c-AblViral oncoprotein E1A, E7, Large T AgViral transcription factor EBNA-5, HCMV IE2

although the precise mechanism(s) of activation is not yet known.pRB may repress the activity of factors such as E2F1 by recruitment of histone

deacetylase (HDAC1) to the promoter. HDAC1 facilitates the formation of nucleosome structures that block access of positive transcription factors to the promoter. In addition, pRB binds a number of co-repressors including RbAp48, RBP1, and C-terminal interacting protein (CtIP), which function by directly repressing transcription or by recruiting HDAC1. However, pRB also represses transcription of promoters that are not sensitive to histone deacetylase activity, by unknown mechanisms. Thus, pRB can actively repress transcription factors, block access of transcription factors to basal transcriptional machinery,or directly inhibit the basal transcriptional machinery.

In addition to regulation of polymerase II transcription, pRB regulates polymerase I genes by binding and inhibiting the ability of an upstream binding factor to bind the ribosomal DNA promoter. pRB binds the general polymerase III transcription factor TFIIIB to regulate genes such as tRNA, 5s RNA, and TATA-box containing genes such as U6. Polymerase III transcription is also regulated by binding of p107 and p130 to the BRF subunit of TFIIIB.

The signaling molecule Raf-1 binds pRB within 30 min of serum stimulation, directly linking mitogenic signaling and cell cycle regulation, which may be one mechanism of transducing Ras cell surface signals to the cell cycle. Inactivation of Ras by a monoclonal antibody in RB1+/+ cells efficiently inhibits DNA synthesis. In RB1−/− cells, inhibition of DNA synthesis is defective. Thus, pRB is required for cell cycle progression downstream of Ras.

4.6 Differentiation by Retinoblastoma ProteinCell culture systems have revealed a role of pRB in determining the fate of the cell. pRB positively regulates terminal adipocyte differentiation of murine embryonic fibroblasts by binding to and activating the CCAAT/enhancer-binding proteins (C/EBPs), a family of transcription factors necessary for adipocyte differentiation. When the cell is induced to differentiate, pRB binds to C/EBP. When U937 cells respond to phorbol 12-myristate 13-acetate by differentiation into monocyte/macrophage lineages, pRB binds NF-IL6, another member of the C/EBP family. Production and maintenance of a terminally differentiated muscle cell phenotype requires binding of pRB to MyoD both in vitro and in vivo. Differentiating human keratinocytes contain pRB/c-Jun complexes that are absent from non-synchronized cycling cells, in part by facilitating binding of c-Jun to an AP-1 consensus site. In bone development, pRB interacts with an osteoblast transcription factor, CBFA1, resulting in synergistic transactivation of an osteoblast-specific reporter. This finding is significant since osteosarcoma is the second most common tumor after retinoblastoma in RB1+/− individuals, and loss of pRB occurs in 60% of sporadic osteosarcomas, suggesting a bone-specific tumor suppressor function of pRB. These culture experiments are consistent with the idea that differentiation involves activation by pRB of genes that are needed for cell type–specific differentiation.

4.7 Genome Stabilization by Retinoblastoma Protein[information on how pRB loss leads to genome instability, Julien Sage 2010 wrote a perspective on RB loss and chromosome instability, and then followed by more articles]

5 RB Family Regulation of Murine Development5.1 RB1 in Murine DevelopmentRB1−/− mice show normal early retinal development up to embryonic days (E)13–15, but then die with abnormalities in neurogenesis and erythropoiesis due to placental failure (Table 2). Wild-type trophoblast rescues RB1−/− embryos until birth, when a muscle defect, previously recognized in partial rescue of RB1−/− embryos, is lethal. The cause of early embryonic lethality is excessive proliferation of trophoblast cells and a severe disruption of the normal labyrinth architecture in the placenta, resulting in decreased vascularization and reduction in placental transportation function. RB1−/− mice that are reconstituted with a functionally normal placenta lack many of the phenotypes of RB1−/− mice and show histologically normal dorsal root ganglia, brain, retina, spinal cord, and liver, but they still die at birth or shortly thereafter with marked defects in skeletal muscle and apoptosis of peripheral nervous system and lens.

Tagged RB1−/− cells in the brains of chimeric embryos exhibited extensive S-phase entry. However, unlike the widespread apoptosis in the germline RB1−/− mice, the majority of the RB1−/− cells in the chimera survived and differentiated into neurons. The developing retina of chimeric embryos showed ectopic mitoses and substantial cell degeneration, while the contribution of RB1−/− cells to the adult retina was much reduced. Therefore, in the CNS, cell-autonomous cell cycle regulation is dependent on functional pRB, but neuronal cell survival and fate may be non-cell-autonomous. In the chimeric CNS, an exception was the RB1−/− Purkinje neurons that were lost. Thus, pRB function may be required in a cell-autonomous manner for cell cycle regulation in most tissues but suppression of apoptosis and differentiation may be either cell-autonomous or non-cell-autonomous, depending on the tissue involved. These

data suggest that the role of pRB in development and differentiation is more important for some cell types than others.

5.2 p107 and p130 in Murine DevelopmentThe importance of the pRB family in differentiation is also demonstrated by p107−/−; p130−/− mice, which are viable until birth and but show deregulated chondrocyte growth and defective endochondral bone formation (reduced rib cage size and shortened limbs). The importance of each pRB family member may be dependent on the level of expression in a particular cell type, and may also depend on the strain of mouse, suggesting the existence of strain-specific modifier genes. Deletion of p130 in Balb/cJ mice resulted in embryonic lethality, while the same deletion in C57BL/6J mice produced viable mice with a reduction in the number of spinal cord and dorsal root ganglia neurons and myocytes, and neural tube and brain apoptosis with increased proliferation.

Balb/cJ and C57BL/6J mice lacking p107 were viable like their counterparts but the Balb/cJ mice displayed impaired growth, reaching only 50% of the weight of their counterparts by 21 days of age. There was also myeloid hyperplasia in the spleen and liver and a twofold acceleration of the cell cycle in p107−/− fibroblasts and myoblasts, suggesting a role for p107 in negatively regulating the overall length of the cell cycle.Tab. 2 RB1 phenotypes in mice.Mouse PhenotypeNo retinoblastomaRB1+/− RB1−/− pituitary and thyroid tumorsRB1−/− Lethal E13-E15, abnormalities in neurogenesis and

erythropoeisis, apoptosis, andinappropriate S-phase entry in CNS, PNS,and lens

RB1−/− with normal placenta Die at birth with marked defects in muscle andlens, no abnormalities in neurogenesis anderythropoeisis

RB1−/−; RB1+/+ chimeric Normal, apoptosis of RB1−/− cells in retinaNesCre1-Cre; RB1 loxP/loxP Apoptosis in lens and PNS but not in CNS,

inappropriate S-phase entry in CNS, PNS, and lensIRBP-Cre; RB1 loxP/loxP Neuroendocrine tumors of the pineal and pituitary

glandp107−/− Normal on C57B/6J, 50% smaller on Balb/cJp130−/− Normal on C57B/6J, die at E11.5 on Balb/cJRB1+/−; p107−/− Retinal dysplasia; RB1−/−; p107−/− pituitary and

thyroid tumorsRB1−/−; p107−/− Lethal E11.5RB1+/−; E2F1−/− Reduced frequency of pituitary and thyroid tumors,

increased life span compared to RB1+/− mouseRB1−/−; E2F1−/− Lethal E17RB1+/−; p53−/− Retinal dysplasia, RB1−/−; p53−/− pituitary and

thyroid tumorRB1+/−; Arf−/− Accelerated pituitary tumor development compared

to RB1+/− mouseRB1−/−; Apaf1−/− No apoptosis in CNS and lens, reduced apoptosis in

PNS and skeletal musclesRB1−/−; Id2−/− Survive to term with no defects in neurogenesis and

hematopoiesis seen in the RB1−/− mice, die at birth with severe reduction in muscle tissue

MMTV-RB_K Dysplasia in mammary tissue, breast cancerPapillomavirus E7 Apoptosis in the retinaViral oncoprotein-induced retinoblastomaSV40 TAg transgene (retina) Retinoblastoma, brain tumorPapillomavirus E7 & E6 transgene (retina) RetinoblastomaPapillomavirus E7, p53−/− RetinoblastomaChimeric knockout retinoblastomaRB1−/−; p107−/− (chimera) RetinoblastomaTargeted RB1−/− knockout retinoblastomaPax6α-Cre; RB1loxP/loxP ; p107−/− RetinoblastomaNesCre1-Cre; RB1loxP/loxP ; p130−/− Maternal inheritance results in retinoblastoma

The expression of p107 and p130 mRNA helps explain some of these data. While p130 is expressed at very low levels throughout embryogenesis, there is a notable increase in expression in developing bone, which would explain the bone phenotype in the p107−/−; p130−/− knockout mice. p107, on the other hand, is expressed highly in the heart, lung, kidney, and intestines but there is no detectable phenotype in these tissues in the p107−/− mice. There is also high expression of p107 in the CNS and liver, which would explain the accelerated apoptosis seen in the liver and CNS in the RB1−/−; p107−/− mice compared to RB1−/− mice.

5.3 E2F in Murine DevelopmentpRB’s ability to block cell growth has always been associated with its ability to block E2F activity. Therefore, it was hypothesized that the loss of E2F in the mouse should have a profound effect on development. Surprisingly, the E2F1 knockout mouse was viable but highly tumor prone and contained some tissue atrophy. This suggested that E2F1 was not vital for proliferation of all cell types, possibly because the other E2Fs can compensate, but is required to block proliferation in some cells while promoting proliferation in other cells. Of particular interest is the enlargement of the thymus and lymph nodes as a result of lack of E2F1. This expansion is a result of an increase in the number of mature CD4/CD8 cells and appears to be due to a lack of apoptosis, consistent with the role of E2F1 in apoptosis. However, E2F1 appears to be an important target of pRB in the regulation of tumorigenesis and development. The loss of E2F1 in the RB1 heterozygous mouse resulted in reduced frequency of pituitary and thyroid tumors and increased life span of the RB1+/−; E2F1−/− mouse compared to RB1+/− mouse. Loss of both RB1 and E2F1 in the RB1−/−; E2F1−/− mouse resulted in embryonic lethality but the mouse lived longer than the RB1−/− mouse (E17 compared with E13.5), suggesting that some functions of pRB during development occur through E2F1. Loss of DP1 leads to embryonic lethality due to failure of extra-embryonic lineage development, suggesting a role for the pRB/E2F pathway in placental development.

6 Retinoblastoma in MiceThe generation of RB1−/− mice was expected to provide information regarding development of retinoblastoma. However, embryonic lethality in RB1−/− mice precluded studies of later development of the retina. Unlike humans, RB1+/− mice do not develop retinoblastoma but instead develop pituitary tumors in which both RB1 alleles are inactivated. The extreme sensitivity of developing human retina to loss of RB1 has stimulated intense efforts to mimic human retinoblastoma in mice, with limited success. The features of the susceptible retinal cell, when defined, may well be the features shared by cancer-susceptible cells in many tissues.

6.1 Viral Oncoprotein-induced RetinoblastomaThe first transgenic mouse model of retinoblastoma arose when the simian virus 40 large and small T antigens (Tag/tag), expressed from the human luteinizing hormone beta subunit (LHβ) promoter in order to produce pituitary tumors, was serendipitously expressed (under the controlregulation of an still-unknown genomic control region) in a specific cell Müller cell subtype of retina with malignant potential. This transgenic line of mice develops focal bilateral retinal tumors that are histologically very similar to human retinoblastoma, because ofcaused by high expression of TAg in the retina. Given its ease of generation, 100% penetrance, and histological similarity to the human disease, this model has been widely used for preclinical therapeutic studies of retinoblastoma. Viral oncoproteins such as HPV E7 and SV40 Tag inactivate pRB when they bind the A/B domain of pRB. TAg binds the active, hypophosphorylated, but not the inactive, hyperphosphorylated form of pRB and also inactivates p107, p130, p53, and other proteins. However, expression of TAg or E7 from the retina-specific promoter interphotoreceptor retinoid-binding protein (IRBP) or opsins does not result in tumors like retinoblastoma. However, IRBP driven HPV E7 results in retinoblastoma in p53−/− mice.

6.2 Chimeric Knockout RetinoblastomaThe retina was normal in both embryonic RB1−/− mice and chimeric RB1−/−; RB1+/+ mice, and RB1−/− cells contributed to most tissues. However, RB1- deficient cells were excluded from the outer and inner nuclear layer of the retina by apoptosis, suggesting that pRB is required during

specific stages of postnatal retinal differentiation. While both the p107−/− and p130−/− mice are viable with no retinal dysplasia, loss of p107 and one copy of pRB in the RB1+/−; p107−/− resulted in retinal dysplasia. Although the RB1−/−; p107−/− mice die at E11.5, preventing evaluation of the developing retina, loss of both p107 and pRB in the retinoblasts of RB1−/−; p107−/− chimeric mice resulted in retinoblastoma. Using expression markers for terminally differentiated retinal cell types, the authors concluded that the retinoblastoma originated in cells committed to the amacrine cell compartment of the inner nuclear layer (INL) but not in cells committed to the outer nuclear layer (ONL).

6.3 Targeted RB1−/− Knockout RetinoblastomaConditional inactivation of RB1 by expression of Cre recombinase in IRBP expressing photoreceptor cells (IRBPCre; RB1loxP/loxP mice) failed to cause retinoblastoma, even when p107 and TP53 were also inactivated. Conditional inactivation of RB1 by expression of Cre from the Pax6α enhancer in p107−/−; RB1loxP/loxP mice resulted in ectopic proliferation of all retinal precursors but only the precursors of horizontal, amacrine, and M¨uüller glia cells survived and terminally differentiated. The Pax6α enhancer is active in peripheral retinal progenitors at E10. Sixty-eight percent of these mice developed tumors in the peripheral retina as early as P8, with larger tumors filling the vitreous at later stages. Markers for amacrine (NeuroD and Math3) and Müller glia (Hes5) were present in the P8 tumor, with few markers for photoreceptor (Crx), bipolar (Chx10), and ganglion cells (Brn3B). P30 tumors expressed markers for amacrine (NeuroD, Math3, Pax6, syntaxin, and calretinin) and M¨uüller glia (Hes5, GFAP, and CRALBP) only.

Conditional inactivation of RB1 by expression of Cre from the nestin promoter (NesCre1) results in mosaic expression of Cre through all layers of the retina with maternal NesCre1 inheritance. Breeding these mice with p130 knockout mice resulted in retinoblastoma tumor formation in five out of five mice, of which four developed bilateral retinoblastoma. The tumors expressed amacrine markers (syntaxin and calretinin) and a few M¨uüller glia cells (GFAP expressing) were also present. However, no tumor stained for the photoreceptor specific gene IRBP, the bipolar cell marker protein kinase C-α (PKC-α), or the retinal ganglion cell marker Brn3b. A potential amacrine cell origin of these mouse tumors is consistent with the earlier mouse model. However, these mouse tumors contain Homer–Wright rosettes but not the classical Flexner–Wintersteiner rosettes pathognomonic of human retinoblastoma, nor any evidence of photoreceptor differentiation.

Examination of the tumor for markers may define some features of the cell of origin, but may be misleading: progenitor cells destined to follow a pathway that is dependent on pRB may adopt a different fate in the absence of pRB. Retinal progenitors go through a series of changes in intrinsic properties that control their competence to make different cell types. If a pRB-related pathway is disturbed, a cell may follow a default pRB-independent pathway, defined by extrinsic cues from their neighbors. This could explain why the majority of human retinoblastomas show characteristics

of photoreceptors (Flexner–Wintersteiner rosettes), while much evidence suggests that they originate in the INL and not in the ONL.

The timing of inactivation of RB1 may play an important role in the development of retinoblastoma. Unlike adult mice, pRB is expressed in all retinal cells at different times during development. Therefore, loss of pRB at different developmental times could result in different retinal phenotypes. This could explain why mice expressing TAg from two different promoters necessary for photoreceptor development have different phenotypes (no tumors with the opsin promoter versus the entire photoreceptor layer developing tumors with the IRBP promoter).

7 RB ProteinpRB and ApoptosisAmong the diverse functions of pRB is the ability to protect cells from apoptosis. RB1−/− embryos die at E13.5 with apoptosis in the CNS, PNS, lens, and other tissues, but rescue of these mice by a normal placenta allows the animals to survive to birth, when a muscle defect is lethal. Similarly, RB1−/−; Id2−/− mice show no defects in neurogenesis and hematopoiesis, but die at birth from severe reduction in muscle tissue. Id2 plays a role in regulating cell death in differentiated neurons, and may also protect against the loss of RB1 in placenta.

Comparison of RB1−/−; p53−/− double knockout mice with RB1−/− mice show that the CNS and lens apoptosis are p53 dependent. However, the apoptosis in the PNS was p53 independent.

Cells in the CNS of the RB1−/−; p53−/− mouse continue to ectopically enter S-phase but do not die. The p53-dependent cell death in the CNS but not the lens is due to hypoxia as a result of defective erythropoiesis. However, conditional knockout of RB1 in the CNS, PNS, and lens in the presence of normal erythropoiesis show similar levels of apoptosis in the PNS and lens compared to the RB1−/− mice, suggesting that the apoptosis in these tissues is a direct result of RB1 loss. The NesCre1 mice with RB1 knockout only in retina show high levels of p53-independent retinal apoptosis in late development.

Deregulated expression of E2F1 in fibroblasts results in S-phase entry followed by apoptosis, which is blocked by expression of high levels of pRB. Apoptosis induced following loss of pRB is dependent on the activity of E2F1, since apoptosis does not occur in the lens of the double knockout RB1−/−; E2F1−/− mice. Additionally, mutant mouse embryos lacking E2F3 and RB1 survive longer than the RB1−/− mice and display a significant reduction in apoptosis and inappropriate S-phase entry.

E2F1 induces cell death in a p53- dependent manner by increasing the stability of the p53 protein. In part, this is due to transcriptional activation of p14ARF (p19ARF in mouse), which promotes the rapid degradation of MDM2 in MDM2-p53 complexes, blocking the degradation of p53 by MDM2. However, p53-dependent apoptosis in the lens of the ARF−/−; RB1−/− mice is only partially suppressed and p53-dependent apoptosis and inappropriate S-phase entry in the CNS are similar to that in RB1−/− mice.

E2F1 also induces transcription of the p53 homolog, p73, which can induce apoptosis. A dominant negative p73 mutant can suppress E2F1-induced apoptosis in cells lacking p53. Additionally, E2F1-induced apoptosis is severely impaired in p73−/− MEFs. Another transcriptional target of E2F1 is APAF1, which plays an important role in stress- and oncogene-induced apoptosis. Levels of APAF1 are increased in RB1−/− mice, and apoptosis in APAF1−/− MEFs is impaired in response to increased E2F1 activity. Analysis of RB1−/−; Apaf1−/− mice shows that APAF1 is absolutely required for the apoptosis in the CNS and lens of the RB1−/− mouse, but only partially rescues the apoptosis in the lens and muscle.

The transactivation domain of E2F1 plays a role in p53-dependent apoptosis but not in p53-independent apoptosis. E2F1 may also play a role in p53- independent apoptosis. Transcriptionally

impaired E2F1 mutants induce apoptosis in Saos2 cells, which lack p53 and pRB, while a DNA-binding defective mutant fails to induce apoptosis. The p53- independent induction of apoptosis by E2F1 can be inhibited by direct binding of pRB to E2F1, but is not inhibited by MDM2. Thus, induction of p53- independent and p53-dependent apoptosis by E2F1 occurs through different mechanisms: p53-dependent apoptosis may rely on relief from repression of promoters and p53-independent apoptosis may involve transcriptional activation by E2F1.

pRB may also protect cells from p53- independent apoptosis induced by ionizing radiation, transforming growth factor β1 (TGF-β1), and γ -interferon (IFN-γ ). Following DNA damage, cells can arrest

in both G1/S and G2/M phases of the cell cycle. pRB is required for the G1/S phase arrest but not for the G2/M phase arrest. This phenomenon is characteristic of pRB alone. The other members of the pRB family are not able to block cells in G1/S following DNA damage.

8 Additional Genomic Changes in Retinoblastoma 8.1 Genomic Gains and Losses (excluding MYCN-amplified retinoblastoma)Although mMutation of both copies of the RB1 gene is usually required for formation initiation of retinoblastoma, with the exception of MYCN retinoblastoma, discussed above. However, all RB1-/- retinoblastoma tumors also have additional genetic alterations required for . There is no evidence that any gene besides RB1 initiates retinoblastoma; however, oThus, other genomic changes may be required for full malignant transformation,. These which have been termed M3-Mn, extending Knudson’s classic M1 and M2 mutational events in the RB1 gene. Karyotypic analysis and comparative genomic hybridization (CGH) show genomic gains and losses in retinoblastoma tumors. The minimal regions most frequently gained are 1q31 (52%), 6p22 (44%), 2p24-25 (30%), and 13q32-34 (12%) and the minimal region most frequently lost is 16q22 (14%). Some evidence suggests that gains at 1q, 2p, and loss of 16q are restricted to more advanced tumors in older children, possibly correlating with tumor progression. Almost unique to retinoblastoma is a specific pattern of 6p gain, i(6p), identified in 60% of retinoblastoma tumors. This marker chromosome results in four copies of genes on chromosome 6p, or

in low-level amplification. The gGains and losses may point to potential oncogenes or tumor suppressor genes necessary for progression to malignant retinoblastoma. Identification of these cancer genes is important as they may form the basis of therapy to prevent the progression of small retinoblastoma tumors.

8.2 Candidate M3-Mn Genes (excluding MYCN-amplified retinoblastoma)Narrowing the region of 6p gain to a minimal 0.6-Mb region of 6p22 using quantitative multiplex polymerase chain reaction (QM-PCR) for sequence-tagged sites (STS) identified RBKIN, a novel human kinesin-like gene, as a potential oncogene in retinoblastoma. RBKIN expression is increased in retinoblastoma compared to normal human retina, and inhibition of RBKIN in retinoblastoma cell lines using antisense oligonucleotidesblocks cell proliferation, consistent with a role for RBKIN as an oncogeneDek and E2F3 as potential oncogenes in retinoblastoma. Chromosome 6p gain is also common in bladder cancer and is associated with an elevated risk of progression.

Cadherin-11 (CDH11) was identified as a potential tumor suppressor gene using LOH and QM-PCR to define the minimal region of 16q22 genomic loss in retinoblastoma. The intact form of CDH11 is either completely lost or decreased, while the variant form is present in 50% of retinoblastoma. CDH11 is a cell adhesion molecule whose loss may promote progression of some RB1−/− retinal cells to malignancy. Unusually low levels of the intact form of CDH11 are present in osteosarcoma, while the variant form was highly expressed in invasive breast cancer and was associated with promotion of invasiveness.

[add KIF14, Syk, miRNAs]

8.3 Causes of Drug Resistance in RetinoblastomaLing and coworkers first described the multidrug resistance (MDR) phenotype in human cancer, due to up-regulation of the MDR1 gene with increased expression of P-glycoprotein. P-glycoprotein expression is increased in response to environmental toxicity in many organisms, and broadly reduces intracellular levels of vinca alkaloids (vincristine, vinblastine), epipodophyllotoxins (etoposide, teniposide), and other natural-product antineoplastic agents by functioning as an ATP-dependent plasma membrane pump that expels drugs from human cancer cells. Platinum compounds are not substrates of the P-glycoprotein drug efflux mechanism.

Expression of MDR1 in retinoblastoma at initial diagnosis and increased expression at relapse after failed treatment may account for frequent chemotherapy failures. It is not possible to correlate P-glycoprotein levels before therapy with the outcome of chemotherapy in intraocular retinoblastoma, since biopsies incur risk of systemic spread. In metastatic retinoblastoma, P-glycoprotein expression before therapy correlates with failure of therapy, and initially undetectable P-glycoprotein correlates with long-term remission.

In order to save eyes without radiation, which increases the lifelong risk of secondary malignancies and other long-term side effects, chemotherapy has become the primary therapy for intraocular retinoblastoma in many centers worldwide. One protocol with excellent success to cure eyes without use of radiation or removal of the eye includes a direct approach to counter P-glycoprotein MDR. High dose cyclosporine A (CSA) is delivered simultaneously with the chemotherapy to block the ability of P-glycoprotein to protect tumor cells from chemotherapy. An international Phase II Clinical Trial of CSA-modified chemotherapy is underway.

Intraocular retinoblastoma that failed chemotherapy without CSA showed increased P-glycoprotein when examined by immunohistochemistry. Retinoblastoma that failed chemotherapy despite administration of CSA did not express P-glycoprotein but expressed instead the Multidrug Resistance Protein (MRP). MRP belongs to the same ATP-dependent membrane transporter superfamily as P-glycoprotein, and transfection of the MRP gene confers a similar broad-spectrum pattern of drug resistance to antineoplastic agents. Although CSA inhibits P-glycoprotein in vitro, there is presently no effective inhibitor of MRP used in humans.

Several chemotherapy drugs induce apoptosis in RB1−/− cells and growth arrest in RB1+/− cells. It is proposed therefore that continued expression of pRB in some tumors may contribute to drug resistance. These drugs might selectively destroy RB1−/− tumors, while pRB expressing adjacent normal cells might be protected by pRB.

9 Importance of Retinoblastoma

The rare eye cancer, retinoblastoma, continues to contribute much more than its ‘‘share’’ to understanding of cancer, biological processes, and use of scientific knowledge to optimize health outcomes. RB1 is the first tumor suppressor gene discovered and revealed the process (search for loss of heterozygosity) that was used to discover other genes underlying human cancer. The RB gene family members are key cell cycle regulators, so that their mutation alters development and deregulates the cell cycle to induce cancer in susceptible cells. Meanwhile, the ongoing search for the retinoblastoma cell of origin has spurred considerable research into normal retinal development. The many ways that RB1 can be damaged and the high mutation rate have forced creative technology development to meet the challenges of clinical diagnostics, much of which has been widely generalized to other genetic disease challenges. At a health service level, it is clearly shown that molecular determination of individual RB1 mutant alleles contributes importantly to health quality at less cost.

Analysis of genetic changes in retinoblastoma subsequent to loss of both RB1 alleles has revealed several potential novel genes involved in tumor progression and resistance to therapy, which will clearly become the targets for potential prevention and therapeutic strategies. Recognition of the key role of the MDR gene in retinoblastoma provided the rational basis for a therapeutic strategy to counter drug resistance that appears to improve the cure rate of intraocular retinoblastoma and that is currently the basis of an international multicenter clinical trial. Although basic studies clearly show an important role of MDR in many cancers, only in retinoblastoma has a therapeutic strategy to alter MDR effect shown positive clinical outcome.

Much more is yet to come. The focus on defining the unique features of the exact cell in developing human retina that is susceptible to cancer by loss of pRB is key to understanding ‘‘cancer stem cells.’’ Retinoblastoma is one of the best examples of human disease indicating that only rare cells at a specific developmental and proliferative stage may be at risk to initiate tumors. And the rarity of retinoblastoma has been turned again to advantage by the world participation of major retinoblastoma treatment centers in an Internet survey to validate a new clinical classification, showing that global cooperation can reach out to future optimal opportunities for all families afflicted with this rare, unique cancer.

See also Cancer Chemotherapy, Theoretical Foundations of; Epigenetic Mechanisms in Tumorigenesis; Intracellular Signaling in Cancer; Growth Factors and Oncogenes in Gastrointestinal Cancers; Oncology, Molecular; Oncogenes.

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