BHS 116.3: Physiology III
Notetaker: Stephanie Cullen
Date: 4/22/13
Page: 1
Lecture 30
Molecular Basis of Cancer
- Non-lethal genetic damage at the heart of carcinogenesis
o Don’t kill the cell
- Multi-step process
o Number of things that have to go on to result in a gene mutation to lead to cancer
formation
- Genes involved include:
o Growth and differentiation promoting (oncogenes)
o Growth and differentiation inhibiting (tumor suppressor genes)
 Normal function is to suppress tumor formation
 Lose that ability when mutated – tumors progress
o Apoptosis genes
o DNA repair genes
 Main function when there is DNA damage is to repair it and prevent tumor
formation and propagation
Genes Involved in the Pathogenesis of Cancer
- All of these genes have a normal function in the cell
o See carcinogenesis when they are mutated only
- Oncogenes
o Ras
 Membrane associated G protein
o Cyclin D
 Transcription role
o CDK4 (cylin dependent kinase)
 Transcription role
- Tumor suppressor genes
o P53 (tp53)
o Rb
 Both affect gene transcription – direct
affects on DNA
- DNA repair genes
o BRCA
 Found in breast cancer
- Apoptotic genes
o BCl-2
Molecular Basis of Cancer
- Normal cell w/ DNA damage from a chemical, radiation,
virus, etc
o Have the tools necessary to repair the gene so
mutations don’t occur if everything in the cell is
working properly
- Could be certain inherited mutations or genes that are not
functioning properly that compound this process
o Have failure of DNA repair
o Damaged DNA can propagate
o Mutation in somatic gene
BHS 116.3: Physiology III
Notetaker: Stephanie Cullen
Date: 4/22/13
Page: 2
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Leads to activation of growth promoting genes (oncogenes), could activate a tumor
suppressor, or a gene that regulates apoptosis
o Combo of unregulated cell proliferation and decreased apoptosis
Tumor proliferation/colonial expansion
Need more blood delivered to the tumor when it is larger than 2mm in order to get blood to the
center of the tumor
o Angiogenesis occurs
Tumor expresses surface proteins that allow protection from the immune system so it won’t be
destroyed by the T cells and B cells
Other mutations allow the tumor to progress and become malignant
o Can invade other tissues – local and through metastasis through the bloodstream
Multi-step process that starts w/ mutation in a specific gene
Oncogenes
- Ras
- Cyclins
- Cyclin-dependent kinases
- Lots of different genes can be mutated to lead to tumor formation
o PDGF-receptor
o TGF
Ras
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Member of a family of small G-proteins (GTP binding) that associates w/ the cytoplasmic surface
of the plasma membrane
o Linked to receptors that are activated usually by growth factors or other signaling
molecules
Plays an important role in mitogenesis induced by growth factors
o Stimulating cell proliferation and growth
Tethered to the cytoplasmic side of the membrane alternating from activated (GTP bound) to
inactivated (GDP bound) states
o Takes binding of the receptor to the ligand to trigger the active form of Ras
Approximately 30% of all human tumors contain a ras mutation
o Most common oncogene abnormality in human tumors
The mutant ras protein is permanently activated because of an inability to hydrolyze GTP into
GDP
o Unable to be inactivated
o Once it binds GTP, the GTP is going to stay bound
o Stimulates its downstream proteins and leads to certain gene transcriptions
Ras Activation
- Ras inactive (bound to receptor)
o Bound to GDP
o Linked to receptor via linking protein
 Not a direct binding
- When a ligand binds to the receptor (1), ras is activated
o GDP dissociates from ras and is replaced by
GTP (2)
o Active Ras recruits RAF-1 (3) and stimulates the
MAP (mitogen activated protein) Kinase
pathway (4)
BHS 116.3: Physiology III
Notetaker: Stephanie Cullen
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Date: 4/22/13
Page: 3
This leads to transcription activity and progression through the cell cycle (5)
 Cell division occurs
The activation is terminated by a GTPase Activating Protein (GAP)
 Stimulates hydrolysis of GTP to GDP – normally
 Mutated ras cannot trigger this hydrolysis
 Constantly stimulating MAP pathway and proliferation and progression
through the cell cycle
Cyclins and Cyclin-Dependent Kinases (CDKs)
- CDK4: interacts w/ cyclin D
- Are other CDKs (CDK2 and CDK1)
- Play roles in the cell cycle in various check points
o Manager of the various stages of the cell cycle and whether or not the cell should
progress to the next step in the cell cycle
Cell Cycle Review
- G1: pre-synthetic growth
- S: DNA synthesis
- G2: pre-mitotic growth
- M: mitosis
- Cell division
- G0: quiescent
o Permanent cells don’t need to divide for a long
period of time
 Cardiac cells
 Neurons
- Check points b/t the major steps
o Restriction point b/t G1 and S phases
o As long as the cells that serve to guard these regions are active, the cell is allowed to
progress to the next step
- CDKs/cyclins play major roles at these check points
Cyclins and CDKs
- Orchestrate the progression of cells through the cell cycle
- Cyclins bind to CDKs (inactive) resulting in their phosphorylation and activation
o They act together as a complex
 Don’t act individually
o Specific cyclins are produced only at specific stages of the cell cycle
- CDKs phosphorylate critical proteins involved in progressing the cell to the next phase of the cell
cycle
o CDKs are constitutively expressed in an inactive form
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Cyclin D/CDK4: progression from G1 to S phase
o Other complexes are active at different steps of the cell cycle
BHS 116.3: Physiology III
Notetaker: Stephanie Cullen
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Date: 4/22/13
Page: 4
Cyclin D and/or CDK4 dysregulation are the most common in
neoplastic growth
o The dysregulation allows progression through the cell
cycle and tumor development
o Number of checks and balances:
 p21 inhibits CDKs by putting a block on the
active CDK to prevent progression through the
cell cycle
o One of the major players is the Rb gene b/c also
involved in the G1 to S progression
Tumor Suppressor Genes
- Rb
- p53
- BRCA-1 and -2 but they are primarily DNA repair genes
o Could include them here since repairing genes results in suppressing tumor formation
Retinoblastoma (Rb) Gene
- Nuclear protein
o Found in all cell types
o Has a normal function
- Hypophosphorylated (few phosphorylation) form is activated
o Prevents cell cycle progression
- Hyperphosphorylated (multiple phosphates added to it) form is inactivated
o Allowing cell cycle to progress
- Plays a role in regulating the cell cycle
o Specifically functions as a brake in preventing cells from entering S from G1 phase
o This is what is occurring in the hypophosphorylated state
Function of Rb Gene
- Hypophosphorylated form:
o Interacts w/ another protein called E2F (transcription
factor)
o When E2F is bound by Rb, other enzymes cluster on it
o Cluster prevents transcription from occurring –
transcriptional block
- Hyperphosphorylated state (inactive form):
o Can’t bind to E2F
o E2F is able to bind DNA w/o other enzymes bound to it
o Transcription occurs
- Multiple phosphorylations do NOT increase activation in this
case – they DECREASE activation (inactive)
Relationship of Cyclins to Rb
- Cyclins play a role in hyperphosphorylation reaction
o Brake on G1 to S is the hypophosphorylated form
- Activation of Cyclin D/CDK4 plays a role in hyperphosphorylating
that Rb
o Inactivates it
o Allows the cell cycle to progress from G1 to S through the
various genes that are triggered by the E2F
BHS 116.3: Physiology III
Notetaker: Stephanie Cullen
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-
Date: 4/22/13
Page: 5
Number of things that stimulate or inhibit this process
o Growth factors stimulate cyclin/CDK activation
o TGF-β and p53 inhibit
o Balance b/t growth stimulating and inhibiting factors plays a role
Mutations in the cyclins that make them constantly active lead to…
o Hyperphosphorylation of the Rb
o Constant progression of cell cycle
Retinoblastoma (tumor itself)
- Believed to arise from uncontrolled growth of neuroepithelial cells in the retina
- Most common malignant eye tumor of childhood
o Diagnosed at an average of 18 months
o 90% diagnosed before patients reach 5 years
- A large number of patients (95%) have no previous family history
o Not a major factor in determining if you have it or not
- Estimated that around 250-500 new cases occur in the United States yearly
- Symptoms
o Leukocoria
 White pupillary reflex or cat’s eye reflex
 Most common presenting sign (56.1% of cases)
o Strabismus as a result of visual loss
 Second most common mode of presentation
o Can cause secondary changes in the eye due to the tumor itself growing
 Glaucoma (tumor growing causes an increase in pressure), retinal detachment
(tumor pulls on the retina), and inflammation secondary to tumor necrosis
- Intraocular stage (leukocoria)
o Pupil opacity
o Can see the reflex
- Glaucomatous stage
o Proptosis (forward projection of the eyeball)
o Tumor pushes the eye forward
- Extraocular stage
o Tumor is growing back from the retina along the optic nerve
Flexner-Wintersteiner Rosettes
- Tumor histology
o Clusters of cuboidal cells arranged around a central lumen
o Very common in Rb tumors (sure sign that it is a Rb tumor)
Genetics of Rb
- Autosomal recessive disorder?
o Not really considered this
o Can really only get 1 copy from the parent
- Involving the Rb gene on chromosome 13
- Familial form (60-70%) or sporadic form (30-40%)
o Familial form: born w/ 1 mutated copy of Rb gene
 All cells end up having the 1 mutated copy (somatic
cells, retinal cells, etc)
 2nd mutation occurs by some environmental insult
 The 2 mutated copies triggers tumor development
BHS 116.3: Physiology III
Notetaker: Stephanie Cullen
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o
Date: 4/22/13
Page: 6
 Recessive in the fact that you need 2 mutated copies
 BUT both mutated copies aren’t from the parent
Sporadic form: born w/ both normal copies of Rb gene
 Retina develops w/ both normal copies
 Mutation of 1 gene occurs in the retinal cell itself
 2nd mutation in the other copy
 Both mutations are caused by an external factor (toxin, UV radiation, Xray radiation, etc)
Need 2 mutant copies for tumor to develop
Treatment
- Intraocular
o Radiation therapy
o Laser photocoagulation
 Smaller tumors
o Chemotherapy
o Cryotherapy
o Enucleation
 Removal of the eye
 May be safest route to keep it from spreading along the optic nerve
- Tumor is usually fatal once it has spread outside the eye and orbit
o Could get into the lymphatic system and metastasize
o Osteosarcoma - Bone is one target
p53
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Acts as a “molecular policeman” preventing propagation of genetically damaged cells
Nuclear protein
Functions by controlling transcription of important genes involved in cell cycle arrest (p21), DNA
repair (GADD45), and apoptosis (bax)
o p21 is one of the biggest inhibitors of CDKs to inhibit cell cycle progression
Most common genetic mutation in human tumors
o Number of different cancers have p53 damage
DNA damage leads to activation of p53
Binds DNA
Stimulates transcription of p21 and GADD45 right away
o p21 inhibits cell cycle progression b/c the DNA is damaged
o GADD45 repairs the damaged DNA before the cycle is
allowed to progress
 If DNA cannot be repaired, apoptosis is triggered
If the DNA has been damaged severely enough, apoptosis may be
triggered right away by stimulating the bax gene
o Won’t even go through the p21 and GADD45 process
If the cell is senescent, it won’t be dividing anymore – it has reached
its mitotic lifespan
o Doesn’t need to trigger activation of these other enzymes
o Can’t propagate the mutated DNA anyway
o Leave the cell alone
All of these processes are lost when p53 is mutated
o Tumor develops
BHS 116.3: Physiology III
Notetaker: Stephanie Cullen
Date: 4/22/13
Page: 7
BRCA-1 and BRCA-2
- DNA repair genes
- Believed to be involved in the regulation of transcription of certain genes
o BRCA-1 involved in regulation of estrogen receptors activity and is a co-activator of
androgen receptor
- Inherited mutations (passed from mother to daughter) lead to a greater susceptibility to breast
cancer and ovarian cancers
o Definitely a genetic link
 Can get cancer w/o the mutation of these genes but the mutation increases the
susceptibility
o BRCA-1 and 2 mutations are found in 10-20% of familial breast cancers (80% in those
w/ multiple affected members) and 3% of all cases
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Normal function:
If damage to the DNA (especially double stranded DNA breaks), the
BRCA-1 and -2 play significant roles in healing that break and
bringing the 2 DNA strands back together, sealing the break, then
allowing normal error-free DNA to progress
Bc1-2
- Mitochondrial protein
- Apoptosis inhibitor (inhibits cell death)
o Favors tumor cell proliferation
- Translocations of Bc1-2 gene associated w/ lymphoma
o Overexpression of Bc1-2 protects lymphocytes from
apoptosis
o Allows for continued proliferation of lymphocytes
Regulation of Apoptosis
- Inhibits cytochrome C
o Cytochrome C stimulates apoptosis by activating a
caspase
o Once the caspases are activated, apoptosis occurs
o So inhibiting cytochrome C blocks apoptosis by
preventing the caspase activation
- Also acts on Apaf-1 (another caspase co-activator)
- Most of the mutations w/ Bcl-2 are overexpression mutations to
really prevents apoptosis
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Normal cells have a balance b/t Bc1-2 (inhibits apoptosis) and Bax genes (stimulates apoptosis)
Increased activation of Bc1-2 favors cell accumulation (tumor formation)
Increased activation of Bax favors apoptosis (cell death)
BHS 116.3: Physiology III
Notetaker: Stephanie Cullen
Date: 4/22/13
Page: 8
Telomeres
- Incomplete replication of chromosome ends (telomeres)
o Whenever chromosomes split, there are telomeres
 There are a finite number of divisions DNA can go through since
they shorten with each split
o Short repeated sequences of DNA
o Usually there is an incomplete break so that the 2 strands are different
lengths
- Telomerase is the enzyme that adds nucleotides to the end of the telomere
o Brings DNA back to normal size so we can get another chromosomal
division at some point
o Allows another round of mitosis
Telomere Length
- As a normal cell ages, its telomere length decreases until it is too short to go
through more divisions
o Due to inactivation of telomerase
o Germ cells and stem cells have highly active telomerase so they
constantly keep those ends at normal length so the cells can divide
many times
o Normal cells: telomerase activity slows w/ each division so the length is
shortened and eventually have growth arrest
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In immortal (cancer) cells, the telomeres are not shortened
o Due to activation of telomerase (re-activation)
 Divisions occur w/o any interruption
o Leads to cancer development
 Cells are constantly dividing to form tumors so need enough telomeres to do that
 Telomerase is highly active
Clicker question: Where in the cell is the Rb protein located?
a. Plasma membrane
a. Ras
b. Nucleus
c. Cytoplasm
d. Mitochondrial matrix