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What are the four phases to the development of malignancy?
- 1. Transformation
- 2. Growth
- 3. Local invasion
- 4. Distant metastasis
What three factors determine a tumors rate of growth?
- 1. Doubling time of the tumor cells.
- 2. Fraction of cells in cell cycle = growth fraction
- 3. Rate of loss of cells
- NOTE: Cell cycle is not shorter in neoplastic cells, just more cells in it.
What are cancer stem cells and where have they been identified?
- Cells able to self-renew, seen in breast cancer and AML.
- Leukemic forms express the gene BMI1 --- represses cell cycle inhibitors: p161NK4a and p14ARF
Name and describe three pathways of tumor spread.
- 1. Direct seeding of body cavity or surface - carcinomas
- 2. Lymphatics - carcinomas
- 3. Hematogenous - sarcomas, liver and lungs most frequently involved
Name the four classes of normal regulatory genes.
- 1. growth promoting protooncogenes - dominant since only one needs to be damaged for transformation to occur
- 2. tumor suppressor genes - recessive since both alleles need to be hit
- 3. genes regulating apoptosis - can be dominant or recessive
- 4. DNA repair genes - usually recessive - both alleles need to be damaged
Name 7 essential alterations for malignant transformation.
- 1. Self-sufficiency in growth signals (ie. don't need external signals for growth)
- 2. Insensitive to inhib signals
- 3. Evasion of apoptosis (often d/t inactivation of p53)
- 4. Defective DNA repair
- 5. limitless replicative potential (maintenance of telomere length and telomerase fxn)
- 6. Sustained angiogenesis - VEGF
- 7. Ability to invade and met
Name the stages of the normal cell cycle.
G0 -- G1 -- S -- G2 --M
What drives cells through the cell cycle?
- Cyclins and cyclin dependent kinases
- CDKs phosporylate proteins, exrpressed constitutively in an inactive form
- Cyclins synthesized during the cycle and rapidly decline after completion.
List the sequence of events in the cell cycle.
- 1. Signal (ex. GFs, integrins)
- 2. MYC, RAS, other genes
- 3. Production of cyclin D -- activation of CDK4
- 4. RB phosphorylation within RB/E2F/DP1 complex (when RB is hypophos it forms a tight inactive complex with trans factor E2F and DP1)
- - RB/E2F/DP1 binds to promoter regions of E2F responsive genes needed for progression of cell cycle - RB recruits histone deacetylase - compacts chromatin
- - when RB is phos, it dissasociates and allows transscription of genes needed for progression ex. cyclin E, DNA polymerases, thymidine kinase etc.
- 5. Cyclin E then activates CDK2 and cell moves from G1 to S (G1-S transition is restriction point) and DNA synthesis occurs
- 6. E2F then mediates transcription of cyclin A which activates CDK2 - complex regulates prophase
- 7. Cyclin B-CDK1 then causes breakdown of nuclear envelope and intiates mitosis
- 8. Exit from mitosis - need inactivation of cyclin B/CDK1
- 9. Return to G1 or to G0
- So - D/4 then E/2 then A/2 then B/1
Name the cell-cycle inhibitors.
- Cip.Kip: p21 (induced by p53), p27 (responds to TGFb)
- INK4/ARF: p16INK4a (binds D/4) promotes inh effects of RB, p14ARF (incr. p53 levels by inh MDM2)
What are the two checkpoints of the cell cycle and how are they controlled?
- 1. G1/S - p53 - needed for p21 which inhibits cell cycle - slows or stops cycle if DNA damage
- 2. G2/M - mediated through p53 dependent and independent mechanisms
- NOTE: ATM senses DNA damage
What are the 5 steps in cell proliferation?
- 1. GF binding
- 2. Activation of GF receptor - signal transduction
- 3. Signal transport via mesenger proteins to nucleus
- 4. Activation of nuclear regulatory factors -- DNA transcription
- 5. Entry to cell cycle --- cell division
What is an oncogene?
Genes that support autonomous cell growth in cancer cells. Protooncogenes are their normal counterparts.
Name the categories of oncogenes.
- 1. GFs
- 2. GF receptors
- 3. Signal transduction proteins
- 4. Nuclear regulatory proteins
- 5. Cell cycle regulators
Give examples of protooncogenes encoding a GF.
- SIS - encodes b chain of PDGF, often same tumors express more of receptor as well - autocrine. But not enough for mal transformation but incr prol contributes to it.
- Id'ed in OSA and astrocytomas
- Also TGFa, HGF and Fibroblast GFs
Give examples and the mechanism of oncogenesis of protooncogenes encoding GFRs.
- GFR are transmembrane proteins with GF binding site extracellular domain and intracellular domain with tyrosine kinase activity, when GF binds the receptor dimerizes and tyrosine phosporylation of signaling proteins.
- Oncogenic versions often have constituent dimerization and become activated without binding of GF
- Gives constant mitogenic signals to cell
- ex. ERB B1 (EGF receptor gene) overexpressed in SCC, ERB B2 in breast cancer, GIST overexpress c-KIT
Name the most common protooncogene encoding signal transducing proteins and how it works.
- RAS - most common oncogene - mutations reduce GTPase activity of RAS proteins
- carcinomas - KRAS, bladder HRAS, hematopoeitic NRAS
- NORMAL RAS: Inactive RAS binds GDP, when activated exchanges for GTP, active RAS recruits RAF-1 and activates MAP kinase pathway which activates nuclear transcription and mitogenesis.
- Usually activation is transient d/t GTPases which return RAS to inactive state with GDP
- Cycling of RAS depends on: 1. nucleotide exchange - GDP to GTP by nucleotide releasing prot recruited by GFR and 2. GTP hydrolysis with GTPases
- GAPs - increase GTPase activity 1000 fold thereby preventing uncontrolled RAS activity
- Mutations in RAS prevent proper GAP activity and stops the GAP break
- RAS can also activate MAP kinase path and AP-1 transcription factor regulating cell cycle
Give an example of a non-receptor tyrosine kinase oncogene.
c-ABL translocated from chromosome 9 to 22 where it fuses with BCR gene - fusion protein has potent tyrosine kinase activity - see in CML and some ALL
What are transcription factors?
- Proteins that enter nucleus, bind DNA and activate or inhibit transcription of adjacent genes
- Ex of oncogenes: MYC, MYB, JUN, FOS
What are the activities of MYC?
- MYC goes to nucleus, binds to DNA and activates transcription of: ornithine decarboxylase, cyclin D2,
- Also have range of other activities: histone acetylation, decr. cell adhesion, incr. cell mobility, incr. prot sythesis, decr proteinase activity
- Needs survival signals to not apoptose after prol though
- MYC mutations seen in burkitt lymphoma, and carcinomas - breast, colon, lung
Give examples of protooncogenes encoding cyclins and CDKs.
- Cyclin D/ CDK4 common
- Cyclin D1 in mantle cell lymphoma
- CDK4 in glioblastoma and sarcomas
- Cyclin E in breast
Name examples of tumor suppressor genes.
- 1. RB
- 2. p53
- 3. APC/b-catenin
- 4. Others: INK4a/ARF locus, TGFb, NF1, FN2, VHL, PTEN, WT-1, Cadheins, KLF6, PTCH
List the key regulators in the cell cycle (ie. one of these needs to be abnormal in cancer).
1. RB, 2. p16INK4a 3. cyclin D, 4. CDK4
Discuss the role of p53.
- p53 is DNA binding protein in nucleus, need mutation in both alleles to have effect
- p53 results in transcription of p21 which arrests G1 before S
- fxn is cell cycle arrest and initiation of apoptosis after DNA damage
- DNA dep kinases and ATM phosphorylate p53 which unfolds then becomes transcription factor and incr. p21
- Has short T1/2 and degraded with ubiquitin mediated proteolysis
- Also induces transcription of GADD45 - encodes protein for DNA repair
- If repair successful then p53 activates MDM2 which degrades p53
- If no repair then p53 activates BAX --- apoptosis (BAX binds to bcl-2 anti-apoptotic -- so initiates apoptosis)
- Summary: p53 phosphorylated by genes that sense DNA damage -- incr p21 (cell cycle arrest), GADD45 (DNA repair) --
- if no repair then BAX, if repair then MDM2 and p53 degraded
What are the two homologues of p53?
P63 and p73
Describe the APC/b-catenin pathway (WNT signalling pathway).
- b-catenin forms complex with TCF in nucleus (transcription factor) and results in upreg of cMyc, cyclin D1 - leading to prol
- APC binds b-catenin in cytoplasm and degrades it
- mutated b-catenin can occur which cannot be inh by APC
- ALSO b-catenin binds to E-cadherin - NB for adhesiveness, so mutations in b-catenin may result in less cell adhessiveness
Discuss INK4a/ARF mutations.
- Melanomas, carcinomas
- lose capacity to block cyclin D/CDK4 so do not prev RB phosphorylation
Discuss TGFb mutations.
- Colon and gastric cancers
- SMAD4 encodes component of TGFb -- mutated in pancreatic cancer, 2 mutated in colon cancer
Discuss NF1 mutations.
NF1 regulates signal transduction via RAS - with mutations RAS trapped in active state
Dicsuss NF2 mutations.
- Also called merlin, protein product is protein 4.1 in RBC cytoskeleton - bindins to actin and CD44
- mutations result in no cell-to-cell junctions and insensitivity to growth arrest signals
Discuss VHL mutations.
Ubiquitin ligase, mutations prevent ubiquitination and degradation of HIF1 --- increased angiogenic GF (VEGF and PDGF)
Causes cell cycle arrest and apoptosis - by incr p27, loss of PTEN - prol
- cell to cell adherence, reduced in many cancers - local invasion and mets
- Can be 2ry decr d/t mutation in b-catenin (b-catenins bind to them and stabalize expression)
- Transcription protein - target genes: TGFb and TGFR
- Incr transcription of p21 independent of p53 so inh cell cycle
- mutation stop cell cycle block by p21
Receptor for hedghod proteins - regulates genes such as TGFb and PDGF-R
List ways cancer cells evade apoptosis.
- 1. BCL-2 overexpression (anti-ap)
- 2. p53 mutations --- decr BAX and/or BIM transcription (both pro-ap)
- 3. MYC - prol. over expression along with incr. BCL-2 cooperate to give cancer
- 4. Incr survival signals - ex. PI3/AKT pathway - often incr. AKT in cancer d/t mutation
Name the three types of DNA repair defects.
- 1. Mismatch repair - spell checker - without it errors slowly accumulate - microsatellite instability is hallmark
- 2. Nucleotide excision repair - uv light causes xlinking of pyrimidine residues - prevents DNA replication, NERs repair these
- 3. Recombination repair - ex. ATM mutation (ATM senses DNA double strand breaks and stops cell cycle via p53), BRCA-1 and 2
- in breast cancer - both involved in DNA repair and in G1/S checkpoint
Explain how BRCA1 and 2 repair DNA.
- 1. DNA break sensed by ATM and CHEK 2 and phosphorylate BRCA1
- 2. BRCA1 migrates to the break site and complexes with Fanconi anemia protein D2
- 3. BRCA2 then migrates to the break site with RAD51 and together they repair the break
How does neovascularization aid in tumor growth?
- 1. Supplies nutrients and oxygen
- 2. New endothelium produces GF (IGF, PDGF) which are also needed for tumor met
Why are tumor vessels leakier?
What are the two most NB tumor-associated angiogenic factors?
- 1. VEGF and 2. bFGF
- What effect does p53 have on tumor angiogenesis?
- Increase transcription of anti-angiogenic thrombospondin 1 and downreg of VEGF and HIF-1 (incr. VEGF prod)
- So no p53 --- no thrombospondin --- increased VEGF
Name angiogenesis inhibitors.
Thrombospondin, p53, angiostatin, endostatin, tumstatin - latter three produced by cleavage of plasminogen and collagens
List and describe the steps of ECM invasion by tumor.
- 1. Detachment - loss of e-cad, sometimes d/t mutant b-catenin
- 2. Attachment to matrix proteins - receptor mediated attachement to laminin and fibronectin, normal epis have high affinity
- receptors on basal surface, carcinomal cells have more and all over -- more receptors, more invasive tumor
- tumor cells also express integrins which are receptors for ECM components
- 3. Degredation of ECM - proteolytic enzymes or induce host cells to make them, serine proteases, cystein proteases, MMP
- MMP2 and 9 cleave type IV collagen in BM and mobilizes VEGF in BM - have angiogenic effects d/t proteins uncovered but
- also antiangiogenic d/t collagen breakdown results in endostatin and tumstatin
- 4. Migration of tumor cells
What is the role of CD44 in metastasis?
CD44 expressed by T cells to home to high end venules, some tumor cells can express and home
How does the stromal microenvironment affect carcinogenesis?
- 1. Cleavage of collagen IV releases VEGF and degredation of laminin-5 results in a fragment that incr. motility
- 2. ECM stores growth factors in inactive forms: PDGF, TGFb, bFGF - paracrine effect
What chromosomal abnormalities are related to carcinogensis?
- 1. Translocations (common) - can results in overexp of protooncogenes d/t removal from reg elements
- ex. MYC gene translocated in burkitt lymphoma, removed from its regulatory sequences and get MYC overexpression
- similarly cyclin D overexpression in mantle cell lymphoma, activation of BCL2 (anti-ap) gene in fol lymphomas
- or can get recombination with new adjacent genes - growth promoting chimeric protein
- ex. phil chrom - fusion of BCR-ABL - hybrid protein which has tyrosine kinase - results in decr. apop, decr GF requirement,
- decr. cell adhesion, activates many pathways - PI3 kinase, RAS, STATs
- 2. Inversions
What is the effect of gene amplication?
- Activation of protooncogenes - may see several hundred copies, microscopically can see dms or HSRs
- ex. amplification of N-MYC in neuroblastoma, ERB-B2 in breast cancer, cyclin D in others
What epigentic changes can result in cancer?
- Can see hypermethylation of pormoter sequences without damage to DNA sequence, can inactivate tumor suppressor genes
- Methylation takes place on CpG islands in DNA
- Have seen this in P14ARF, p16INK4a, BRCA-1, VHL and MLH1 (mismatch repair gene)
NOTE: most cancer involve mutations of several oncogenes and loss of two or more tumor suppressors
What is the difference between gatekeeper and caretaker genes?
- Gatekeepers - ongogenes and tumor sup genes that control tumor growth
- Caretakers - do not directly control tumor growth but affect genomic stability
For sporadic cancers what two pathways are usually disrupted?
- 1. RB - INK4, Cyclin D, assoc kinases, RB
- 2. p53 - p14ARF, MDM2, p53
List the steps involved in chemical carcinogenesis.
- 1. iniation - permanent DNA damage - but carcinogen induced DNA damage does not always lead to initiation since can be repaired
- 2. Promoter - can induce tumor in initiated cells but not normal cells, do not affect DNA directly, reversible
- application of a promoter lead to prol and clonal exp of initiated (mutated) cells
List the major carcinogens.
- 1. Direct acting:
- Alkylating agents
- Acylating agents
- 2. Pro-carcinogens - need metabolic activation
- Polycyclic heterocyclic aromatic hydrocarbons
- Aromatic amines, amides, azo dyes
- Natural plant and microbial prodcuts - aflatoxin, griseofulvin, cycasin, etc.
- Others: nitrsamine, amides, vinyl chloride, chrominum, fungiides etc.
List some promoters of chemical carcinogens.
cigarette smoke, alcohol, viruses, hormones, bile salts
What are most commonly mutated with UV radiation?
p53 and RAS - get formation of pyrimidine dimers in DNA - usually repaired by NER pathway:
Name the steps of the NER pathway.
- 1. recognition of DNA lesion
- 2. Incision of strand on both sides
- 3. Removal of damaged region
- 4. synthesis of patch
- 5. ligation
Name some DNA viruses which are carcinogenic.
Adenovirues, fibroma in rabbits, BPV
Name some RNA viruses which are carcinogenic.
FeLV, FIV, EIA, Papillomavirus
Explain carcinogenesis of papillomavirus.
- viral DNA inserts, results in E6/E7 overexpression - overcome cell cycle inh.
- often see p53 mutations
Explain carcinogenesis of Helicobacter pylori.
Dz strains have CagA (pathogenicity island) - CagA injected into host cells, also VacA (other virulence gene)
List some tumor antigens:
- 1. Products of mutated oncogenes/tumor suppressor genes
- 2. Products of other mutated genes
- 3. Overexpressed proteins - may normally be in such small amts that there is no self-tolerance
- 4. virus products
- 5. oncofetal proteins - expressed on fetal tissues and by cancer cells but not adult cells
- ex. AFP, CEA
- 6. Altered cell surface glycolipids/proteins
- 7. CD antigens
List the cells involved in tumor immunity.
CD8 Tcells, NK, Macs - NK and T cells produce IFNg which activate macs - ROS killing etc.
List ways tumor cells evade the immune system.
- 1. Selection of antigen-negative variants (survival of the fittest)
- 2. Loss or reduced MHC1 escape CTL
- 3. Lack of costim
- 4. Immunosuppression - ex. secrete TGFb
- 5. Antigen masking - ex. by glycocalyx molecules
- 6. Apoptosis of CTL - express FasL
What cytokines are responsible for cancer cachexia?
- TNF, IL1, IFNg
- Also proteolyis-inducing factor - increases catabolism of fat and mm (not cytokine)