Ref Robbins 7/e p300; , Harrison 17/e p499; 9/e p290 Tumor suppressor genes are the genes whose products down regulate the cell cycle. RB Gene: Governor of the Cell Cycle It is useful to begin with the retinoblastoma gene (RB), the first tumor suppressor gene to be discovered and, as it happens, a prototypical representative. As with many advances in medicine, the discovery of tumor suppressor genes was accomplished by the study of a rare disease--in this case, retinoblastoma, an uncommon childhood tumor. Approximately 60% of retinoblastomas are sporadic, and the remaining ones are familial, the predisposition to develop the tumor being transmitted as an autosomal dom- inant trait. To account for the sporadic and familial occur- rence of an identical tumor, Knudson, in 1974, proposed his now famous two-hit hypothesis, which in molecular terms can be stated as follows: * Two mutations (hits) are required to produce retinoblas- toma. These involve the RB gene, which has been mapped to chromosomal locus 13q14. Both of the normal alleles of the RB locus must be inactivated (hence the two hits) for the development of retinoblastoma (Fig. 5-21). * In familial cases, children inherit one defective copy of the RB gene in the germ line; the other copy is normal. brakes to cellular proliferation Rb gene is a tumor suppressor gene whereas My ,fos and Ra's are all example of proto oncogene Retinoblastoma develops when the normal RB gene is lost in retinoblasts as a result of somatic mutation. Because in retinoblastoma families only a single somatic mutation is required for expression of the disease, the familial transmission follows an autosomal dominant inheritance pattern. * In sporadic cases, both normal RB alleles are lost by somatic mutation in one of the retinoblasts. The end result is the same: a retinal cell that has lost both of the normal copies of the RB gene becomes cancerous. Although the loss of normal RB genes initially was discovered in retinoblastomas, it is now evident that homo- zygous loss of this gene is a fairly common feature of several tumors, including breast cancer, small cell cancer of the lung, and bladder cancer. Patients with familial retinoblastoma also are at greatly increased risk for development of osteosarcomas and some soft tissue sarcoma The RB gene product is a DNA-binding protein that is expressed in every cell type examined, where it exists in an active hypophosphorylated state and an inactive hyperphosphor- ylated state. The impoance of Rb lies in its regulation of the G1/S checkpoint, the poal through which cells must pass before DNA replication commences. As background for an understanding of how tumor sup- pressors function, it is useful to briefly revisit the cell cycle: In embryos, cell divisions proceed at an amazing clip, with DNA replication beginning immediately after mitosis ends. As development proceeds, however, two gaps are incorpo- rated into the cell cycle: gap 1 (G1) between mitosis (M) and DNA replication (S), and gap 2 (G2) between DNA replica- tion (S) and mitosis (M) (Fig. 5-20). Although each phase of the cell cycle circuitry is monitored carefully, the transi- tion from G1 to S is believed to be an extremely impoant checkpoint in the cell cycle "clock." Once cells cross the G1 checkpoint they can pause the cell cycle for a time, but they are obligated to complete mitosis. In G1, however, cells can remove themselves entirely from the cell cycle, either tem- porarily (quiescence, or G0) or permanently (senescence). Indeed, during development, as cells become terminally differentiated, they exit the cell cycle and enter G0. Cells in G0 remain there until external cues, such as mitogenic sig- naling, push them back into the cell cycle. In G1, therefore, diverse signals are integrated to determine whether the cell should progress through the cell cycle, or exit the cell cycle and differentiate, and Rb is a key hub integrating external mitogenic and differentiation signals to make this decision. To appreciate this crucial role of Rb in the cell cycle, it is helpful to review the mechanisms that enforce the G1/S transition. * The initiation of DNA replication (S phase) requires the activity of cyclin E/CDK2 complexes, and expression of cyclin E is dependent on the E2F family of transcription factors. Early in G1, Rb is in its hypophosphorylated active form, and it binds to and inhibits the E2F family of transcription factors, preventing transcription of cyclin E. Hypophosphorylated Rb blocks E2F-mediated transcription in at least two ways (Fig. 5-22). First, it sequesters E2F, preventing it from interacting with other transcriptional activators. Second, Rb recruits chromatin remodeling proteins, such as histone deacetylases and histone methyltransferases, which bind to the promoters of E2F-responsive genes such as cyclin E. These enzymes modify chromatin at the promoters to make DNA insen- sitive to transcription factors. This situation is changed on mitogenic signaling. Growth factor signaling leads to cyclin D expression and activa- tion of cyclin D-CDK4/6 complexes. These complexes phosphorylate Rb, inactivating the protein and releasing E2F to induce target genes such as cyclin E. Expression of cyclin E then stimulates DNA replication and pro- gression through the cell cycle. When the cells enter S phase, they are committed to divide without additional growth factor stimulation. During the ensuing M phase, the phosphate groups are removed from Rb by cellular phosphatases, regenerating the hypophosphorylated form of Rb. * E2F is not the sole target of Rb. The versatile Rb protein binds to a variety of other transcription factors that regulate cell differentiation. For example, Rb stimulates myocyte-, adipocyte-, melanocyte-, and macrophage- specific transcription factors. Thus, the Rb pathway couples control of cell cycle progression at G0-G1 with differentiation, which may explain how differentiation is associated with exit from the cell cycle. In view of the centrality of Rb to the control of the cell cycle, an interesting question is why RB is not mutated in every cancer. In fact, mutations in other genes that control Rb phosphorylation can mimic the effect of RB loss; such genes are mutated in many cancers that seem to have normal RB genes. For example, mutational activation of CDK4 or overexpression of cyclin D ors cell proliferation by facil- itating Rb phosphorylation and inactivation. Indeed, cyclin D is overexpressed in many tumors because of gene ampli- fication or translocation. Mutational inactivation of CDKIs also would drive the cell cycle by unregulated activation of cyclins and CDKs. As mentioned earlier, the CDKN2A gene is an extremely common target of deletion or muta- tional inactivation in human tumors. The emerging paradigm is that loss of normal cell cycle control is central to malignant transformation and that at least one of the four key regulators of the cell cycle (CDKN2A, cyclin D, CDK4, Rb) is mutated in most human cancers. Fuhermore, the transforming proteins of several oncogenic human DNA viruses act, in pa, by neutralizing the growth inhibi- tory activities of Rb. For example, the human papillomavi- rus (HPV) E7 protein binds to the hypophosphorylated form of Rb, preventing it from inhibiting the E2F transcrip- tion factors. Thus, Rb is functionally deleted, leading to uncontrolled growth