Molecular basis of cancer

Question 1

Tissue homeostasis

Answer 1

in a normal tissue there is a balance of cell:

• surival
• growth and division
• differentiation
• death

Question 2

LOs

ØReview key concepts which underpin the molecular basis of cancer-tissue homeostasis and the role of oncogenes and tumour suppressor genes (TSG)

ØConsider genetic/epigenetic features of tumourigenesis and the role of tumour viruses

ØStages of tumour development – multi-step model of cancer and cancer stem cells

ØExplore the biology of metastasis

Question 3

Normal cell proliferation process

Answer 3

1. The binding of a growth factor to its specific receptor
2. Transient and limited activation of the growth factor receptor
3. Activates several signal-transducing proteins on the inner leaflet of the plasma membrane
4. Transmission of the transduced signal across the cytosol to the nucleus via second messengers or by a cascade of signal transduction molecules
5. Induction and activation of nuclear regulatory factors that initiate DNA transcription
6. Entry and progression of the cell into the cell cycle, ultimately resulting in cell division

Question 4

In order to multiply successfully, normal cells require extracellular signals that drive:

Answer 4

• cell-cycle progression
• cell growth

Question 5

What is thr cell cycle clock?

Answer 5

• denotes a molecular circuitry operating in a cell nucleus that processes and integrates a variety of afferent signals from within and from outside of the cell.
• It then decides whether or not the cell should enter into the active cell cycle or retreat into a non-proliferating state
• if active porliferation is decided on, the circuitry needs to prgram the complex sequence of biochemical changes to enable it to double its contents and divide

Question 6

Checkpoint control system of the cell cycle

Answer 6

• •The sequential events of the cell cycle are directed by a distinct cell cycle control system.
• Regulated by internal and external controls.
• Has multiple checkpoints where the cell cycle stops until a go ahead signal is received
• signals indicate if key cellular processes have been completed correctly
• 2 types of regulatory proteins are involed in cell cycle control: cyclins and cyclin dependant kinases.
• Activity of CDK rises and falls with changes in conc of cyclin partner.

Question 7

G1/ S -cyclins

S -cyclins

M -cyclins

G1 -cyclins

Answer 7

G1/ S -cyclins - activate CDKs in late G1 and help commit to cell cycle entry. Their levels fall in S phase

S -cyclins- bind CDKs soon after progression through start and help stimulate chromosome duplication. Remain at high levels until mitosis, and these cyclins contribute ti the control of some early mitotic events.

M -cyclins- activate cdks that stimulate entry into mitosis at the G2/ M transition. M-cyclin levels fall in mid-mitosis.

G1 -cyclins- govern the G1/s cyclins

Question 8

Pairing of cyclins to their CDKs

D type

E

A

B

Answer 8

D- (D1, D2 and D3) bind CDK4 and 6

E- (E1 and E2) bind CDK2

A- (A1 and A2) bind CDK2 and CDC2

B- (B1 and B2) bind CDC2

Question 9

Fluctuation of cyclins during cell cycle

focus on D

Answer 9

• Fluctuations are generally tightly coordinated with the schedule of advances through the various cell cycle phases
• However, for D-type cyclins, extracellular signals, (especially growth factors) strongly influence their levels
• While cyclin D1 is present in other cell cycle phases besides G1, following the G1/S transition it is exported from the nucleus into the cytoplasm, where it can no longer influence cell cycle progression

Question 10

Name some extracellular signals that induce D type cyclin expression (mitogens???)

Answer 10

RANK receptor D1

Prolactin r D1

oestrogen r D1

HER2 D1

FSH r D2

Question 11

Cell cycle dependant phosphorylation of Rb

Answer 11

The phosphorylation state of Rb is closely controlled. it is indicated by the red line

during M/G1 transition, virtually all existing phosphate groups are stripped off Rb, leaving it unphosphorylated.

Progressing through G1 phase, a phosphate group is added to 1/14 phosphorylation sites on Rb- hypophosphorylated state.

At the restriction point (R)- cyclin E-CDK2 complexes phosphorylate Rb on >12 sites- hyperphosphorylated state.

Throughout the rest of cell cycle remains constant until M phase

Question 12

Functional consequences of phosphorylation

Answer 12

Unphosphorylated Rb:

• Binds TFs called E2Fs
• therefore prevents E2F mediated transcriptional activation of many genes whose products (e.g., DNA polymerase are required for DNA synthesis)

Phosphorylated Rb:

Cyclin D-CDK4/6 kinase activity phosphorylates Rb in mid G1

Complete phosphorylation inactivates Rb causing E2F to disassociate E2Fs, allowng them to turn on genes required for transition to S for DNA synthesis, irreversibly comitting the cell to DNA synthesis.

Deregulation of the cell cycle and genome maintenance pathways can cause cancer.

Mutations that promote upregulated passage from G to S phase are oncogenic

Question 13

What kind of cancer does Rb loss of function lead to?

Answer 13

childhood retinoblastoma

cancers later in life- breast, bladder, carcinoma of lung

Question 14

CDK inhibitors

p27Kip1

p16INK4A

Answer 14

• p27Kip1 blocks cyclin A–CDK2 function by obstructing the ATP-binding site in the catalytic cleft of the CDK.
• Inhibitors of the INK4 class, such as p16INK4A, bind to CDK6 and CDK4. These CDK inhibitors distort the cyclin-binding site of CDK6, reducing its affinity for D-type cyclins. At the same time, they distort the ATP-binding site and thereby compromise catalytic activity.

Question 15

The INK4b-ARF-INK4a locus encodes for 3 tumnour supressor genes

Answer 15

• encodes for 3 so is prone to mutations causing oncogenic changes
• p16 acts as a TSG- inhibiting G1 cyclin D-CDK4/6 kinase activity are common
• p14ARF encodes a key activator controlling stability of the tumour suppresor p53

therefore a mutation in the INK4b-ARF-INK4a locus can simultaneously inhibit Rb and p53 pathways.

Question 16

p53- guardian of the genome

Answer 16

the p53 gene is the most common target for genomic alteration in human tumours

p53 acts a molecular guardian by preventing the propagation of genetically damaged cells

acts mainly at G1/S and G2/M checkpoints

homozygous loss of p53 occurs in virtually every type of cancer

Question 17

p53 inhibits neoplastic transformation by interlocking mechanisms

Answer 17

1. quiescense- activation of temporary cell cycle arrest
2. senescence- induction of permanent cell cycle arrest
3. apoptosis- triggering of programmed cell death

Question 18

Tumour viruses general

Answer 18

Cancers that are attributable to infections have a greater incidence than any individual type of cancer worldwide

11 viruses have been deemed carcinogenic agents

most common causes:

1. H. pylori
2. HPV
3. hep B
4. hep C
5. EBV

Knowing that these viruses can cause cancer means that we can take preventative measures to stop them –> HPV vaccines

Question 19

Tumour viruses interactions with Rb and p53

Answer 19

often seek to inactivate Rb and p53 tumour supressors as they stand in the way of a viruses ultimate goal: efficient mulitiplication in tissues of infected cells.

most viruses parasitize host cell DNA replication machinery in order to replicate their own genomes. this ,machinery is only available in late G1 and S phases of the cell cycle, therefore deactivate Rb.

these quiescent cells then enter S phase

cells activate p53 in response to excessive p53

Question 20

HPV causing cancer

Answer 20

•Infect replicating cells in the cervical epithelium and block the normally occurring exit from the active cell cycle that takes place as these cells differentiate.

Oncogenic potential of HPV can be related to the products of two viral genes, E6 and E7

The E6 protein binds to and mediates the degradation of p53 and BAX (pro-apoptotic member of the BCL2 family) and it activates telomerase

The E7 protein binds to the Rb protein and displaces the E2F transcription factors that are normally sequestered by Rb, promoting progression through the cell cycle

Question 21

Human T cell leukaemia virus type 1

Answer 21

T cell laeukaemia/ lymphoma that is endemic in japan and caribbean

retorvirus from retrovidae family

Viral DNA gets integrated into the host chromosome and the tax protein inactivates p53

Also inactivates p16/INK4a, and activates cyclin D, thus dysregulating the cell cycle

Question 22

Critical step in tumourigenisis- viral integration

Answer 22

can occur either due to:

recombinant events (dsDNA) or as part of the normal life cycle of an RNA retrovirus such as HTLV-1 or RSV

multiple mechanisms:

• chronic expression of proteins interfering with TSG functions
• expressoin of viral proto-oncogenes (RSV)
• Transcriptional activation of endogenous proto-oncogenes (e.g., myc)
• inactivation of endogenous TSG

Question 24

cancer can result from the expression of mutant forms of 7 types/groups of proteins

Answer 24

1. Extra-and intra-cellular signaling molecules
2. Signal receptors
3. Signal-transducing proteins
4. Transcription factors
5. Cell-cycle control proteins, which function to restrain cell proliferation
6. DNA-repair proteins
7. Apoptotic proteins – tumour suppressors that promote apoptosis and oncoproteins that promote cell survival

Question 25

mutations that lead to cancer are either:

Answer 25

a gain of function: ocogene e.g,. overexpression of transmembrane mitogen receptor

a loss of function: in a TSG e.g., p53

Question 26

2 hit hypothesis

Answer 26

takes 2 dysruptions to some genes for cancer effects to come into play

works only for autosomal dominant genes???

e.g., Rb

Question 27

epigenetic changes also play a part in cancer

Question 28

not all TSG mutations are recessive

Answer 28

e.g., BRCA 1 and BRCA 2

Question 29

Typs of activating mutations

Answer 29

plus mutations that stabilize the protein e.g., myc

Question 30

cancer mutations; multi-step hypothesis

intratumour heterogeneicity

Answer 30

carcinogenesis is a multistep process resulting from the accumulation of multiple mutations:

these mutations accumulate independantly in different clonal cells, generating subclones with varying abilities to grow, invade, metastasize and resist or respond to therapy

over time tumours not only increase in size but become more aggressive and acquire a greater malignant potential (tumour progression)

Question 31

Clonal selection

Answer 31

• population of clones where one acquires an advantagous mutation, allowing it to grow/ survive better
• subsequent mutations in progeny can cause descendents to grow more uncontrollably and form a small benign tumour
• third mutations allow tumour to escape the constraints imposed by the tumour microenvironment and outgrow the others to form a mass of cells, each of which has all 3 gentic changes
• fourth allows cell to escape into bloodstream and establish colonies at other sites

cells in a given tumour should have at least some genetic alterations in common

cancer incidence shouold increase with age - it takes time for mutations to accrew

Question 33

Adenomatosis Polyposis Coli in colon cancer

mutation effects

Answer 33

a protein that supresses the Wnt proliferative pathway as part of the destruction complex

in an epithelial cell–> uncontrolled cell proliferation to form a localised polyp of benign tumour cells

dysregulation of ras (activates other proteins- GTPase) and loss of TSG TP53 generates a malignant cell

cell progeny invade basement membrane that surrounds the tissue but does not invade blood vessels

eventual invasion of blood vessels

addittional mutations let it leave blood vessels

Question 34

unlocking limitless replicative potential (cancer hallmark)

Answer 34

Tumour cells have unrestricted proliferative capacity, avoiding cellular senescence and mitotic catastrophe.

Two barriers prevent cultured cells from replicating indefinitely in culture:

a) Senescence involves cells existing long-term in a non-growing but viable state
b) Crisis involves the apoptotic death of cells

Normal cells can divide 60-70 times. their limit is reached due to progressive shortening of telomeres at the ends of chromosomes

in cells that have disabled checkpoints– DNA repair pathways inappropriately activated– massice chromosomal instability, mitotic instability and cell death

chromosmal instability leads to breakage-fusion-bridge cycles and aneuploidy + acquisition of mutant alleles

cells can escape mitotic catastrophe by activating hTERT telomerase, elongating telomeric DNA

Question 36

Ability to invade and metastasize

Answer 36

Tumour metastases are the cause of the vast majority of cancer deaths and depend on processes that are intrinsic to the cell or are initiated by signals from the tissue environment

Question 37

invasion-metastasis cascade (7 steps)

Answer 37

1. Primary tumour formation.
2. Localized invasiveness enables in situ carcinoma cells to breach the basement membrane.
3. Intravasation into either lymphatic or blood microvessels.
4. Transportion via the general circulation, to distant anatomical sites.
5. Tumour cells may become trapped, subsequently extravasate, and form dormant micrometastases.
6. May eventually acquire the ability to colonize the tissue in which they have landed, enabling them to form a macroscopic metastasis.
7. The last step – colonization – seems to be the most inefficient of all.

Question 38

Sequence of events in invasion of epithelial basement membranes by tumour cells

Answer 38

A.Downregulation of E-cadherin expression (through a variety of pathways) reduces the ability of cells to adhere to each other and facilitates their detachment from the primary tumour and their advance into the surrounding tissues.

B.Tumour cells degrade the extracellular matrix (ECM) by secreting proteolytic enzymes themselves or inducing stromal cells like fibroblasts and inflammatory cells to elaborate proteases.

C.Attachment to novel ECM components

D.Degradation of basement membrane and tumour cell migration follow.

Question 39

intravasation

Answer 39

requires help from stromal cells- in this case macrophages

triad established between carcinoma cells, macrophages and endothelial cells

carcinoma cells have to ecxpress mena actin-cytoskeleton-regulating protein

Question 40

extravasation

Answer 40

1. A metastasizing cell is trapped physically in a capillary.
2. Within minutes, a large number of platelets become attached to the cancer cell, forming a microthrombus.
3. The cancer cell pushes aside an endothelial cell on one wall of the capillary, thereby achieving direct contact with the underlying capillary basement membrane.
4. Within a day, the microthrombus is dissolved by the proteases that are responsible for dissolving clots.
5. The cancer cell begins to proliferate in the lumen of the capillary.
6. Within several days, sometimes sooner, the cancer cells break through the capillary basement membrane and invade the surrounding tissue parenchyma.

Question 41

Ability to invade and metastasize

Answer 41

Once in the circulation, tumour cells are vulnerable to destruction.

So, tumour cells tend to aggregate in clumps.

This is favored by homotypic adhesions among tumour cells as well as heterotypic adhesion between tumour cells and blood cells, particularly platelets. Formation of platelet-tumour aggregates may enhance tumour cell survival and implantability.

Organ tropism may be due to the following mechanisms:

1. Tumour cells may have adhesion molecules whose ligands are expressed preferentially on the endothelial cells of the target organ
2. target tissue may be a non-permissive environment e.g., skeletal muscles rarely the site of metastases

Question 42

Epithelial mesenchymal transition

Answer 42

process in which epithelial cells disaggregate and exhibit dramatic shape changes

process by which cells acquire a “de-differentiated” phenotype

transitioning epithelial cells lose polarity and intercellular contacts then gain mesenchymal properties:

• increased migratory capacity
• increased contractility
• increased production of extracellular matrix proteins

Question 43

• There are key steps in growth and proliferation
• Viruses can initiate cancer by subverting checkpoints
• Genetic changes responsible for cancer act to increase activity of proto-oncogenes or reduce the activity of TSG
• Cancer is not the result of mutations in a single gene – multi-step model of cancer and cancer stem cell hypothesis
• Many cancer cells do not obey normal rules of limited proliferative capacity – role of telomerase in aggressive tumours
• Metastasis is associated with many cancer related deaths – complex multi-stage process involving subpopulation of tumour cells capable of EMT