Cell Cycle & Cancer_Cirillo_Notes.pdf

Cell Cycle and Cancer Lisa Ann Cirillo, PhD Foundations of Medicine Learning Objectives: By the end of this session, learners should be able...

Cell Cycle and Cancer Lisa Ann Cirillo, PhD Foundations of Medicine Learning Objectives: By the end of this session, learners should be able to… 1. Define the phases of the eukaryotic cell-cycle. 2. Explain the basic signaling events that regulate each cell-cycle phase and checkpoint. 3. Describe how cell proliferation must generate two "daughter" cells that are genetically identical, with the alternative being cancer. 4. Distinguish the 5 categories of genes, including one example of each, that when mutated cause cancer. 5. List the principles by which cancer cells may be therapeutically targeted. References:  Molecular Cell Biology, Lodish et al., 9e, Chapter 19, Sections 19.1, 19.3 - 19.5 and 19.7 on cell cycle control Learning Session Outline: I. The Cell Cycle A. Intro to the cell cycle 1. Cell cycle – sequence of events enabling DNA replication and cell division 2. There are 1013 (ten trillion) cells in the human body! 3. Selected cells proliferate to renew us, on average, once every 7 years. 4. examples: a. neurons & muscle cells (myocytes) never divide (i.e., they are senescent). b. liver cells (hepatocytes) divide 1x yearly. c. gut, skin, bone marrow cells divide 2x daily. 5. Mission of the cell-cycle: generate 2 daughter cells that are genetically identical. a. alternative = disease or cancer B. Phases of the cell cycle 1. Interphase – portion of the cell cycle when the cell is not actively dividing (G1  S  G2) a. Gap 1 (G1): cell grows and produces proteins needed for DNA replication i. Cell has choices during this phase: a. Senescence (G0) b. Differentiation (G0) c. Apoptosis (cell death) d. Proliferation  cell-cycle b. Synthesis (S): synthesis of chromatin, DNA is replicated i. 2N DNA  4N DNA ii. Histones increase 2x c. Gap 2 (G2): cell grows and prepares for cell division (M-phase) i. Centrosome duplicates ii. Additional proteins needed for mitosis are synthesized 2. Mitosis (M) - cell divides into two identical daughter cells 3. Resting phase (G0) – cell is fully differentiated and no longer dividing; cell has exited the cell cycle 4. Duration of each phase: a. S, G2 & M ~ constant b. G1 is variable, hence, total generation time (Tg) is variable. Foundations of Medicine Cell Cycle & Cancer Lisa Cirillo, PhD  C. Proteins regulate the cell cycle 1. External factors a. Hormones b. Cytokines c. Growth factors i. Activators: FGFs, IGFs, Wnts ii. Inhibitors: TGFβ 2. Internal factors a. Early response factors: myc, fos, jun b. Delayed response factors i. cyclins + CDKs = cyclin-dependent protein kinase (CDK) heterodimers D. Cyclin-dependent protein kinase heterodimers 1. Kinases are enzymes that reversibly attach negatively-charged phosphate PO4-- groups to a wide variety of target proteins (i.e. substrates) that regulate the cell-cycle. a. PO4-- groups are attached to serine [S], threonine [T] & tyrosine [Y] residues. b. Cells have thousands of protein phosphorylation sites. 2. Phosphorylation induces a conformational change that regulates a protein’s activity, its ability to bind other factors, etc. 3. Cyclin-dependent kinase – protein kinase which must be activated by a cyclin; functions to regulate the cell cycle E. What Happens when the Cell-Cycle is Induced? (during G 1) 1. First: a. p27, a CDK inhibitor, decreases & stays low. b. cyclin-D production increases & remains high 2. Then cyclins E, A, B sequentially & transiently increase F. Cell Cycle Regulation 1. The cell cycle is regulated at by 4 Cyclin/CDK-4 Partners a. G1 entry: Cyclin-D/CDK-4 b. G1 checkpoint: Cyclin-E / CDK-2 i. Entry into S phase – is the cell ready to divide? a. Big enough? b. Enough energy/reserves? c. DNA damage? c. S phase: Cyclin-A / CDK-2 d. G2 checkpoint: Cyclin-B / CDK-1 i. Is all DNA replicated? ii. DNA damage? e. M checkpoint: Cyclin-B / CDK-1 i. All chromosomes attached to spindle? 2. G1 entry: early G1 regulated by cyclin-D/CDK-4 a. Growth factor  myc  cyclin-D b. cyclin-D binds CDK-4 c. cyclin-D/CDK-4 phosphorylates retinoblastoma (Rb) d. pRb releases E2F (a transcription factor) e. E2F activates genes for cyclin-E & cyclin-A 3. G1 checkpoint: the G1 “Restriction” Checkpoint is regulated by cyclin-E/CDK-2 a. Cyclin-E binds CDK2 b. Cyclin-E/CDK2 phosphorylates target proteins breaches R, the Restriction checkpoint, at G1/S to initiate S-phase. c. The restriction checkpoint is guarded by p53 i. p53 inhibits the cell-cycle at G1/S by inducing p21, which binds to and inhibits CDK2 ii. p53 is mutated, causing loss-of-function, in ~½ of cancers. Foundations of Medicine Cell Cycle & Cancer Lisa Cirillo, PhD  4. S-phase: transit throughout S-phase is regulated by cyclin-A/CDK-2 a. Cyclin-A binds CDK2. b. Cyclin-A/CDK-2 phosphorylates proteins in DNA replication complexes. 5. G2 checkpoint is regulated by cyclin B/CDK-1 a. De-phosphorylation of cyclin-B/CDK-1 is required to activate mitosis. b. cdc25, a phosphatase, de-phosphorylates the CDK1 subunit of the cyclinB/CDK1 heterodimer. c. cyclinB/CDK1  nucleus  multiple phosphorylations i. Nuclear envelope breakdown ii. Assembly of mitotic spindle iii. Metaphase arrest 6. M checkpoint is regulated by cyclin=B/CDK-1 a. Cyclin-B/CDK-1 triggers its own destruction through activation of APC/C (anaphase promoting complex/cyclosome) i. Attachment of chromosomes to spindle apparatus required b. APC/C-mediated destruction of cyclin-B/CDK1 via ubiquination pushes cells into anaphase c. APC/C-mediated destruction of proteins holding sister chromatids together (cohesins) results in movement of chromosomes to opposite poles of cell II. Cancer is a Genetic Disease A. Characteristics and Causation 1. Characteristics: a. Point mutations b. Chromosome re-arrangement, loss, gain c. Mutant cell clones expand  tumor d.  cellular differentiation,  proliferation e. Pathogenic invasiveness 2. Causation: a. As cells age, mutations accumulate  malignancy B. 5 gene groups that are mutated in cancer 1. Proto-oncogenes a. Proto-oncogenes (~75) are genes that encode: i. cell membrane receptors for growth factors a. EGFR  non-small cell lung carcinoma b. Src (a tyrosine kinase receptor)  sarcoma; colon cancer ii. cytoplasmic & transcription factors a. Ras (a GTPase)  25% of all human cancers b. Myc (a transcription factor)  many cancers including Burkitt’s lymphoma iii. cyclins/CDKs a. Cyclin-D b. When mutated, proto-oncogenes become “oncogenes”. c. Oncogenes cause pathological activation of the encoded protein  cell-cycle activation  tumor. 2. Tumor suppressor genes normally suppress the cell-cycle a. Proteins that normally suppress the cell-cycle are tumor suppressors. b. Major tumor suppressors are: i. p21: inhibits CDK-4 & CDK-2 ii. p16: also inhibits CDK-4; mutated in many cancers iii. p53: induces a. Apoptosis b. Gene encoding p21 iv. Rb: binds & inhibits E2F-1 Foundations of Medicine Cell Cycle & Cancer Lisa Cirillo, PhD  v. BRCA-1 & BRCA-2 repair broken DNA c. Mutations inactivate tumor suppressors. d. Clinical correlation: BRCA1-2 mutations increase the risk of cancer more than other factors 3. Genes that regulate apoptosis (programmed cell death) a. Apoptosis = programmed cell death (PCD) b. Apoptosis occurs normally, and, it is necessary. c. It occurs in both embryos, & in adults. i. 50-70 billion cells apoptose daily. ii. Defective cells (i.e. tumor) are removed. d. Steps in apoptosis: i. Macrophages secrete TNF (tumor necrosis factor)  TNF receptor. ii. Pro-apoptotic (Bax) & anti-apoptotic (Bcl-2) factors become un-balanced. iii. Bax ↑ in the outer mitochondrial membrane. iv. Cytochrome-C leaks out, activating caspase (a protease). v. Caspase fragments chromatin  cellular disruption. e. Mutated “apoptosis genes” cause cancer. i. B Cell Lymphoma is caused by mutated BCL-2 gene. a. Normally, Bcl-2 inhibits apoptosis. b. When mutated, Bcl-2 over-actively inhibits apoptosis  tumor. 4. Genes that regulate cell senescence (telomerase) a. Cells have a finite lifespan, because at each cell division telomere DNA is lost from the end of each chromosome. i. The telomere DNA subunit is TTAGGG, repeating up to ~15,000 bp. ii. At each cell division, 50-100 bp are lost. b. After 60-70 cell divisions, chromosome shorten, causing replicative senescence. c. How telomere length regulates senescence is poorly understood. d. The enzyme telomerase maintains telomere length in ESCs & germ cells. But, telomerase is normally inactive in somatic cells. e. When telomerase is mutated in somatic cells, it becomes ‘constitutively’ active, causing cancer due to immortalization of these cells. 5. Genes that repair DNA a. The cell-cycle machinery makes ~1 mistake/billion bp. b. Normally, the cell-cycle stops, pending repair, if DNA is damaged. c. If DNA repair genes (such as the BRCAs) are mutated, carcinogenic cells emerge, resulting in tumors. i. Examples: colo-rectal cancer, breast cancer 6. Clinical correlation: cancer prevention and treatment a. Cancer Prevention: clinical testing i. DNA testing to detect BRCA mutations. ii. Serum testing for bladder cancer antigens, prostate-specific antigen (PSA), etc. b. (Most) cancer treatments are non-specific i. Goal: specific treatments to: a. target metastasis (drugs that inhibit metallo-proteases) b. target angiogenesis (prevent new BV growth) c. target specific molecules 1. Rituxan  CD20 in B cell lymphoma and leukemia (CLL) 2. Herceptin  HER2 receptor in breast cancer 3. Venetoclax  BCL2 in leukemia (CLL and AML) Foundations of Medicine Cell Cycle & Cancer Lisa Cirillo, PhD