Cancer A disease of the genome – Genomic instability A disease of the cell cycle – Checkpoint failures A disease of the aged Benign vs. Malign What makes a cancer cell so special? – Interaction with basal lamina – Interactions with other cells – Blood Supply

DysplasticACFEarlyAdenomaIntermediateAdenomaLateAdenomaCarcinomaMetastasisNormalEpithelium APC other changes? DCCDPC4/JV18? K-RasMMRDeficiency

Cancer Progression Example: Colorectal Cancer APC mutations initiate the neoplastic process. Patients with FAP inherit APC mutations and develop numerous dysplastic aberrant crypt foci ACF, which then progress MMR deficiency speeds up this process K-RAS is an oncogene that requires only one genetic event for its activation. The other specific genes indicated are tumor suppressor genes that require two genetic events (one in each allele) for their inactivation Chromosome 18q21 may contain several different tumor suppressor genes involved in colorectal neoplasia, with DCC, DPC4, and JV18–1 genes proposed as candidates

Cancer The pathway to carcinogenesis consists of 4-7 rate-limiting events. Tumor development proceeds by a process analogous to Darwinian evolution. The multiple lines of cellular defense may explain why cancer is not more frequent during an average human lifetime. If you live long enough, you will develop cancer. Cell, Vol. 100, 57–70, January, 2000

Cancer The pathway to carcinogenesis consists of 4-7 rate- limiting events. – Recently, as little as 2: activation of a pro-growth (c-myc) factor and a survival factor (Bcl-X L ) or suppression of a proapoptotic p53 or p19ARF Tumor development proceeds by a process analogous to Darwinian evolution. The multiple lines of cellular defense and the maintainence of DNA fidelity may explain why cancer is not more frequent during an average human lifetime. If you live long enough, you will develop cancer. Cell, Vol. 100, 57–70, January, 2000

General Charactistics of Cancer All cancers must acquire several of the same six hallmark capabilities Means and order of acquisition vary significantly. The catalyst of acquiring these is Genomic instability.

Cancer: A disease of the Genome Genomic Stability Given the standard mutation rate in dividing cells, coincident with the fidelity of DNA replication, the time it would take to achieve a sufficiently mutated state for cancer would far surpass the human lifespan. – A normal error frequency of 1 base-pair change in roughly 10 9 base pairs for each cell generation (1 in a billion). – A single gene that encodes an average-sized protein (~10 3 base pairs) suffers a mutation once in about 10 6 cell generations. – This number is roughly consistent with the evolutionary estimate - one mutation appears in an average gene in the germ line every 200,000 years.

Genomic Instability DNA damage/mutation Apoptosis Mitosis Arrest Repair Attempt Extensive damage Correction Normal Cell Damage to Apoptotic Pathway Mitosis Damage Cancer Cell Inability to Sense Damage Damaged Repair Mechanisms Inability to arrest Rapid accumulation of genomic damage. leads to accumulation of more key cancer cell characteristics Perpetuation of errors to daughter cells. DNA damage/mutation

Environmental Causes of Genomic Instability and Aneuploidy Oncogenic Viruses Chemical carcinogens Ionizing radiation Loss of checkpoints + circumvention of apoptosis = genomic instability

Cell, Vol. 100, 57–70, January, 2000 General Charactistics of Cancer All cancers must acquire the same six hallmark capabilities Means and order of acquisition vary significantly. The catalyst of acquiring these is Genomic instability.

Characteristics of Tumor Cells Genome Instability Given the normal mutation rate in dividing cells, the time it would take to achieve a sufficiently mutated state for cancer would far surpass the human lifespan. A normal error frequency of 1 base-pair change in roughly 10 9 base pairs for each cell generation (1 in 1,000,000,000, or one in a billion). A single gene that encodes an average-sized protein (~10 3 base pairs) suffers a mutation once in about 10 6 cell generations. This number is roughly consistent with the evolutionary estimate - one mutation appears in an average gene in the germ line every 200,000 years.

Cancer: A Disease of the Genome Genomic Stability Given the standard mutation rate in dividing cells, coincident with the fidelity of DNA replication, the time it would take to achieve a sufficiently mutated state for cancer would far surpass the human lifespan – A normal error frequency of 1 base-pair change in roughly 10 9 base pairs for each cell generation (1 in a billion) – A single gene that encodes an average-sized protein (~10 3 base pairs) suffers a mutation once in about 10 6 cell generations – This number is roughly consistent with the evolutionary estimate – one mutation appears in an average gene in the germ line every 200,000 years.

How is Fidelity Normally Maintained? Protein level: Wobble Effect. Genetic level: Distinct DNA repair mechanisms. – DNA polymerase exonuclease activity – post-replicative recognition, excision, and repair of mismatches Other strategy Affecting stability: 5% exons Extensive introns

Genomic Instability DNA damage/mutation Apoptosis Mitosis Arrest RepairAttempt Extensivedamage Correction Normal Cell Damage to ApoptoticPathway Mitosis Damage Cancer Cell Inability to Sense Damage DamagedRepairMechanisms Inability to arrest Rapid accumulation of genomic damage. leads to accumulation of more key cancer cell characteristics Perpetuation of errors to daughter cells.

DNA Damage Arrest Normal Cell Cancer Cell Repair Damage Apoptosis Mitosis Damaged Repair Mechanism Inability To Sense Damage Inability to Arrest DNA Damage Damage to Apoptotic Pathway Mitosis Rapid accumulation of genetic damage Perpetuation of Errors to Daughter Cells Genomic Instability DNA Damage

Aneuploidy Euploidy: Having a chromosome number that is an exact multiple of the monoploid number. Aneuploidy: Having a chromosome number that is not an exact multiple of the usually haploid number. Develops from defects in the process of chromosome segregation. Most benign tumors are diploid. All malignant tumors are aneuploid; this is one of the identifying characteristics of malignant tumor cells. Is required for cell immortalization; it is a critical rate-limiting step of tumorigenesis – therefore, it develops early in the progression. Caused by the same factors which cause genomic instability.

Causes of Genomic Instability and Aneuploidy Oncogenic Viruses Chemical carcinogens Ionizing radiation Spontaneous occurrence in p53-/- cell lines derived from Li-Fraumeni patients (germline mutation in the p53 gene)

Once genomic instability is in effect, cancer cells then follow a quicker path towards the accumulation of necessary mutations required to reach full-blown carcinogenesis.

Cell 2000 Jan 7;100(1):57-70

Acquired Characteristics of Tumor Cells 1.Self-Sufficiency in Growth Signals 2.Insensitivity to Antigrowth Signals 3.Evading Apoptosis 4.Limitless Replicative Potential (Immortalization) 5.Sustained Angiogenesis 6.Tissue Invasion and Metastasis

Characteristics of Tumor Cells I: Self-Sufficiency in Growth Signals many oncogenes mimick normal growth signaling. 3 strategies: – 1: Alteration of extracellular growth signals (Autocrine stimulation) PDGF (platelet-derived growth factor) TGF  (tumor growth factor alpha) – 2: Signal Transducer alteration A: wild-type GF receptors are overexpressed: hypersensitivity – EGF-R/erbB B: Structural alteration of receptors can result in ligand-independent signaling – EGF receptor lacking most of the cytoplasmic domain fire constitutively – 3: Intracellular circuits RAS - 25% of human tumors, constitutive mutants Cell, Vol. 100, 57–70, January, 2000

II: Insensitivity to Antigrowth Signals Normal tissue: – Antiproliferative signals operate to maintain cellular quiescence and tissue homeostasis. Soluble growth inhibitors Immobilized inhibitors embedded – the extracellular matrix – the surfaces of nearby cells – These growth-inhibitory signals, like their positively acting counterparts, are received by transmembrane cell surface receptors coupled to intracellular signaling circuits.

E Cadhedrin CAMs antigrowth signals  -catenin Lef/Tcf transcription factor N-CAM Wilms tumor, neuroblastoma, and small cell lung cancer Epithelial Cancers antigrowth signals Growth Inhibitory Signal via binding Extracellular Matrix Integrins

Apoptotic signals Apoptotic Signals via detachment

III: Evading Apoptosis: future lecture Bcl-2: Oncogene that doesn’t promote growth, but inhibits death. usually found mutated in conjunction with c-myc, which is a powerful activator of the cell cycle. p53: 50% of human tumors have this gene mutated. Decoy death receptors etc.

DR-4/5 TRAIL DcR TRAIL Decoy Receptor Can still bind ligand, but unable to transduce signal DD

IV: Limitless Replicative Potential: Immortalization Even cells which are free to divide at their whim eventually stop growing. This is due to telomere shortening. How does a cell arrive at immortality?

growthincultureSenescence Loss of Rb or p53 pathways (mutagens, primary tumor cells) ImmortalizationCrisis Massive cell death, karyotypic disarray associated with end-to-end fusion of chromosomes (telomeres no longer protect ends) 1 in 10 7 cells emerges, and is now able to perpetually grow. Pathway to Cellular Immoralization ALT mechanism: maintains telomeres through recombination- based interchromosomal exchanges of sequence information Telomerase is activated or Telomerase expression artificial Ras expression Inhibition of p16 OR p53 p16 OR p53 telomere shortening bp/cycle

growthincultureSenescence Loss of Rb or p53 pathways (mutagens, primary tumor cells) ImmortalizationCrisis Massive cell death, karyotypic disarray associated with end-to-end fusion of chromosomes (telomeres no longer protect ends) 1 in 10 7 cells emerges, and is now able to perpetually grow. Pathway to Cellular Immoralization ALT mechanism: maintains telomeres through recombination- based interchromosomal exchanges of sequence information Telomerase is activated or Telomerase expression artificial Ras expression Inhibition of p16 OR p53 p16 OR p53 telomere shortening bp/cycle

HDACs & HATS: Chromatin Remodelling Overview B

Pathways to Cellular Immortalization Growth In culture Senescence Many divisions CRISISImmortalization Ectopic Ras expression Inhibition of p16 or p53 Massive cell death; karyotypic disarray associated with end-to-end fusion of chromosomes (telomeres no longer protect ends) 1 in 10 7 cells Emerges And is now able to Perpetually grow Telomerase is Activated ALT Mechanism: Maintains Telomeres through recombination- based interchromasomal exchanges of sequence information Telomerase expression Loss of Rb / p53 pathways

V: Sustained Angiogenesis (growth of new blood vessels) All cells, normal and cancer alike, must reside within 100 µm of a capillary blood vessel. A tumor which fails to activate angiogenesis can only grow to ~2mm in diameter.

5. Sustained Angiogenesis All cells, normal and cancer alike, must reside within 100  m of a capillary blood vessel. A tumor which fails to activate angiogenesis can only grow to ~2mm in diameter

Simplistic model of Tumor Angiogenesis endothelial cells

VI: Tissue Invasion and Metastasis 90% of human cancer deaths are due to metastatic tumors. Invasion and metastasis are exceedingly complex processes, and their genetic and biochemical determinants remain incompletely understood.

Molecular Defects in Cancer: Two Paradigms Tumor Suppressors Loss of function p53 p16 p53 p16 Rb pTEN p27 etc. etc. Oncogenes Gain of function c-myc Ras Cyclin D CDK4 Bcl-2 Survivin Oncogenic Viral Proteins Each protein is resopnsible in evading one or more of the core six characteristics necessary for tumorigenesis of the core six characteristics necessary for tumorigenesis

Fighting Cancer

Chemotherapy The idea behind chemotherapy is simple: kill cells which are proliferating. Chemotherpeutic agents induce apoptosis (specific to the cycling cells) and necrosis (nonspecific and therefore less effective) The Failure of Chemotherapy – unfortunately, many cancers have damaged apoptotic response pathways, leading to ineffective treatment – 50% of cancers have a mutation in p53, which is crucial for apoptotic response. – Many normal cells proliferate at the same rate (if not faster) than tumor cells – Many tumor cells don’t necessarily grow faster as they simply just don’t die.

Chemotherapy The idea behind chemotherapy is simple: kill cells that are proliferating through inducing death pathways. Chemotherapeutic agents induce apoptosis (specific to the cycling cells) and necrosis (nonspecific and therefore less effective) The failure of chemotherapy: – unfortunately, many cancers have damaged apoptotic response pathways, leading to ineffective treatment. – 50% of cancers have mutated p53, crucial for the apoptotic response. – Many normal cells proliferate at the same rate (if not faster) than tumor cells – Many tumor cells don’t necessarily grow faster as they simply just don’t die – Strategy: Reactivate death pathways, then hit them with a toxic stress (synergy between chemotherapy and a sensitizing agent).

Targeting Apoptosis in Cancer Therapy Cancer can be distilled to two fundamental genetic lesions: – excessive proliferation – constitutively active survival signaling pathway To compensate, pro-growth signals are also capable of inducing pro-apoptotic signals (c-myc) – Provides a failsafe mechanism to offset oncogenic capacity – irony: creates a pressure for tumor cell selection.

Myc GrowthApoptosis Lesion 1Lesion 2 current chemotherapeutic approaches Novel approaches Strategy: Exploit cancer’s inactivated apoptotic pathways