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Robert A. Weinberg The Biology of Cancer First Edition pRb and Control of the Cell Cycle Clock Nam Deuk Kim, Ph.D. Copyright © Garland Science1 2007 2 Summary The central governor of growth and proliferation 3 Figure 8.1 The Biology of Cancer (© Garland Science 2007) 1. External signals influence a cell’s decision to enter into the active cell cycle The mammalian cell cycle 4 Figure 8.3a The Biology of Cancer (© Garland Science 2007) Examples of checkpoints in the cell cycle 5 Figure 8.4 The Biology of Cancer (© Garland Science 2007) 2. Cells make decisions about growth and quiescence during a specific period in the G1 phase Responsiveness to extracellular signals during the cell cycle 6 Figure 8.6 The Biology of Cancer (© Garland Science 2007) 3. Cyclins and cyclin-dependent kinases constitute the core components of the cell cycle clock Cyclin-CDK complexes 7 Figure 8.7 The Biology of Cancer (© Garland Science 2007) Pairing of cyclins with cyclin-dependent kinases *CDC2 = CDK1 8 Figure 8.8 The Biology of Cancer (© Garland Science 2007) Cell cycle-dependent fluctuations in cyclin B levels 9 Figure 8.9 The Biology of Cancer (© Garland Science 2007) Fluctuation of cyclin levels during the cell cycle 10 Figure 8.10 The Biology of Cancer (© Garland Science 2007) Control of cyclin D1 levels: Cyclin D1 was discovered as a protein whose levels are strongly induced by exposure of macrophages to the mitogen CSF-1 (colony-stimulating factor-1). 11 Figure 8.11a The Biology of Cancer (© Garland Science 2007) Control of cyclin D1 levels: The control of cyclin D1 levels by extracellular mitogens can be explained, in part, by a signal-transduction cascade that leads from growth factor receptors, one of a number of factors that modulate the transcription of the cyclin D1 gene in the nucleus. As indicated, a number of other signaling cascades converge on the promoted of this gene. 12 Figure 8.11b The Biology of Cancer (© Garland Science 2007) 13 Table 8.1 The Biology of Cancer (© Garland Science 2007) Control of cyclin levels during the cell cycle 14 Figure 8.12 The Biology of Cancer (© Garland Science 2007) 4. Cyclin-CDK complexes are also regulated by CDK inhibitors Actions of CDK inhibitors Figure 8.13a The Biology of Cancer (© Garland Science 2007) 15 The complex between p27Kip1 and cyclin A-CDK2, derived by X-ray crystallography. 16 Figure 8.13b The Biology of Cancer (© Garland Science 2007) Inhibitors of the INK4 class, such as p16INK4A, bind to CDK6. This CDK inhibitor distort the cyclin-binding site of CDK6, reducing its affinity for D-type cyclins. 17 Figure 8.13c The Biology of Cancer (© Garland Science 2007) Control of cell cycle progression by TGF-β TGF-β 18 Figure 8.14a The Biology of Cancer (© Garland Science 2007) When TGF-β is applied to human keratinocytes, it evokes a dramatic 30-fold induction of p15 mRNA synthesis as demonstrated here by RNA (Northern) blotting analysis. These cells were exposed to TGF-β for the time periods (in hours) indicated. 19 Figure 8.14b The Biology of Cancer (© Garland Science 2007) Control of cell cycle advance by extracellular signals. receptor tyrosine kinase pathway p15INK4B 20 Figure 8.15a The Biology of Cancer (© Garland Science 2007) Suppression of p27 function by Akt/PKB in human breast cancers. • At low grade (less advanced) tumor A: p27 in the nucleus (>50% nuclear) with less pAkt/PKB increased survival • At low grade tumor B (less advanced) tumor B: p27 in the nucleus (≤ N + C) with higher pAkt/PKB decreased survival 21 Figure 8.16b The Biology of Cancer (© Garland Science 2007) 5. Viral oncoproteins reveal how pRb blocks advance through the cell cycle Cell cycledependent phosphorylation of pRb 22 Figure 8.19 The Biology of Cancer (© Garland Science 2007) 6. pRb is deployed by the cell cycle clock to serve as a guardian of the restriction-point gate. Control of the restriction-point transition by mitogens. 23 Figure 8.22 The Biology of Cancer (© Garland Science 2007) 7. E2F transcription factors enable pRb to implement growth-versus-quiescence decisions. E2Fs and their interactions. 24 Figure 8.23a The Biology of Cancer (© Garland Science 2007) The E2F transcription factors bind DNA as heterodimeric complexes with DP partners: E2F4-DP2 complex to the DNA double helix. 25 Figure 8.23c The Biology of Cancer (© Garland Science 2007) The E2Fs constitute a family of at least seven distinct proteins. • E2Fs 1, 2 and 3a: transcriptional activators • E2F3b, E2F4, E2F5: transcriptional repressors • E2F6, E2F7: poorly resolved 26 Figure 8.23d The Biology of Cancer (© Garland Science 2007) Modification of chromatin by pocket proteins 27 Figure 8.24a The Biology of Cancer (© Garland Science 2007) When the E2Fs are not complexed with pRb (or its p107 and p130 cousins), they attract histone acetylases (red), which place chromatin in a configuration that I conductive for transcription. 28 Figure 8.24b The Biology of Cancer (© Garland Science 2007) Positive-feedback loops and the irreversibility of cell cycle advance. The irreversibility of certain key steps in cell cycle progression and the rapidity of their execution is ensured, in part, by the activation of certain positive-feedback loops. 29 Figure 8.25a The Biology of Cancer (© Garland Science 2007) Positive-feedback loops and the irreversibility of cell cycle advance. 30 Figure 8.25b The Biology of Cancer (© Garland Science 2007) 8. A variety of mitogenic signaling pathways control the phosphorylation state of pRb • Growth factors growth factor receptors Ras cyclin D1 and E inactivation of pRb activation of E2Fs S-phase entrance 31 Figure 8.26 The Biology of Cancer (© Garland Science 2007) Countervailing controls on cyclin D1 levels. The Ras signaling pathway influences cyclin D1 levels in at least three major ways. 1. AP-1 (Fos-Jun) pathway 2. PI3K pathway 3. Ras pathway A fourth minor pathway: 1. GSK-3β pathway (dashed line) 32 9. The Myc oncoprotein perturbs the decision to phosphorylate pRb and thereby deregulates control of cell cycle progression. The Myc transcription factor: • Myc belongs to a family of bHLH (basic helix-loop-helix) transcription factors. • Myc-Max: promote transcription active proliferation • Mad-Max: repress transcription of most target genes increased differentiation 33 Figure 8.27 The Biology of Cancer (© Garland Science 2007) Actions of Myc on the cell cycle clock: • Myc-Max: induce expression of the growth-promoting proteins cyclin D2 and CDK4 promote advance through early G1. • By increasing the expression of Cul1 (which is responsible for degrading the p27 CDK inhibitor) as well as E2Fs 1, 2, and 3, Myc favors advance into S phase. • In addition, Myc, acting with its Miz1 partner, is able to repress expression of the p15, p21, and p27 CDK inhibitors; once again, effects are felt both in early/mid and in late G1. Figure 8.28 The Biology of Cancer (© Garland Science 2007) 34 Powers of the Myc oncoprotein: The wide-ranging effects of the Myc protein are illustrated by an experiment in which the Myc protein has been fused to the estrogen receptor (ER) protein (blue). In the absence of ER ligands, such as estrogen or tamoxifen, the Myc-ER protein is trapped in the cytoplasm (through association with heat shock proteins, not shown). When estrogen or tamoxifen ligands of the ER (small purple ball) are added to cells, the Myc-ER protein migrates into the nucleus, associates with Max, and activates Myc target genes within minutes. Such activation, when induced in serum-starved cells in the G0 phase, enables them to enter the active cell cycle and advance all the way through G1 into the S phase. 35 Figure 8.29 The Biology of Cancer (© Garland Science 2007) 10. TGF-β prevents phosphorylation of pRb and thereby blocks cell cycle progression. • TGF-β: a major growth-inhibitory signal that normal cells, especially epithelial cells, must learn to evade in order to become cancer cells. • Many types of cancer cells must evade TGF-β-imposed growth inhibition if they are to thrive. • TGF-β phosphorylation of Smad3 complex with Smad4 and Miz1 induce expression of p15 and p21 (weakly) • TGF-β dispaches Smad3 to form a complex with E2F4/5/p107 represses expression of Myc gene • TGF-β succeeds in inducing expression of the two CDK inhibitors -p15 and p21and thereby shuts down cell cycle progression in the early/mid G1 phase of the cell cycle. Figure 8.31 The Biology of Cancer (© Garland Science 2007) Countervailing actions of TGF-β and Myc. 36 11. Control of pRb function is perturbed in most if not all human cancer. 37 Table 8.3 The Biology of Cancer (© Garland Science 2007) 38 Table 8.4 The Biology of Cancer (© Garland Science 2007) Perturbation of the R-point transition in human tumors. • The decision to advance through the R-point transition (yellow) can be perturbed in a variety of ways in human tumors. • [Brown color]: favor advance through the R point • [Blue color]: block this advance 39 Figure 8.35 The Biology of Cancer (© Garland Science 2007) Amplification of the cyclin D1 gene. • Cyclin D1 gene (CCND1) in the cells of a head-and-neck squamous cell carcinoma (HNSCC). • CCND1 is amplified to various extents. • About one-third of all of these tumors, leading to corresponding increases in cyclin D1 expression and resulting loss of proper control of pRb phosphorylation. 40 Figure 8.36 The Biology of Cancer (© Garland Science 2007) 12. Synopsis and prospects Cyclin E and breast cancer progression. • This Kaplan-Meier plot presents the clinical progression of disease of women with stage III breast cancer. • Plotted is the fraction of patients (ordinate) who are still alive at the indicated times after initial diagnosis (abscissa). 41 Figure 8.38 The Biology of Cancer (© Garland Science 2007)