Lecture 13 - Hallmarks and resistance to apoptosis

Question 1

Key concepts of cell proliferation regulation

Answer 1

• Normal cells divide only when they receive an extracellular signal - using growth factors or mitogens
• The signal is relayed to the nucleus by a signal transduction cascade ─ ie the mitogenic signalling pathway

Mitogenic signalling activates protein translation and biosynthetic pathways required for energy, anabolic metabolism, and organelle production and triggers entry into the cell division cycle

Question 2

What inhibits cell proliferation

Answer 2

Strong cell-cell contacts and other readouts of intact tissues such as matrix stiffness inhibit proliferation (contact inhibition of growth)

Question 3

Autotransphosphorylation: what is it, what does it do, and where can it be seen?

Answer 3

The name given when one protomer phosphorylates another protomer which then phosphorylates it again

Causes activation of the dimer (?)

In GF binding to growth factor receptors

Question 4

What is a hallmark to cancer growth regarding growth factors?

Answer 4

The transition from paracrine signalling from growth factors (ligand dependent) to ligand independent firing from he receptor (possibly occurring as the cancer cells promote growth themselves)

Question 5

Ras: what is it, what does it do, what can it be substituted for, and what is its role in cancer?

Answer 5

Small globular GTPases (~20kDa) that bind to GTP/GDP for active/inactive forms

When activated, it exposes an effector loop which allows lots of downstream signalling (Raf, PI3K, Ral GEF, etc)

Can be substituted for serum (source of growth factors) - they are growth factors

Improper activation of Ras may lead to cancer formation - one hotspot is glycine-12 in Ras, substituted for valine, leaving Ras almost permanently bound to GTP as the glycine side arm should stretch into the GTP binding pocket and allow binding for GAPs to enhance GTP hydrolysis, but valine does not cause this to occur - growth constantly stimulated (?)

Question 6

Activation/inactivation of Ras

Answer 6

Like most GTPases, they get activated by guanine exchange factor (GEF) swapping a bound GDP for a GTP, and gets inactivated as they do their intrinsic GTP hydrolase activity, which is further enhanced by binding to a GTPase activating protein (GAP)

Question 7

SH2: what is it, what does it do, and what is its role in cancer?

Answer 7

Src homology 2, a domain in proteins that has docking sites for binding of -SH3-SOS-Ras

Allows SH3 to bind which allows SOS, a GEF, to activate Ras

Question 8

MAPK: what is it, what is the typical sequence,

Answer 8

An ancient, highly conserved signalling module comprising sequential phosphorylation of a cascade of protein kinases (amplifies the signal)

• Extracellular signal activates a mitogen-activated protein kinase kinase kinase (MAPKKK) through Ser/Thr phosphorylation
• Which activates a MAP kinase kinase (MAPKK) through Ser/Thr phosphorylation
• Which activates a MAP kinase (MAPK) through Tyr/Thr phosphorylation
• MAPK then goes to activate cell differentiation etc etc through Ser/Thr phosphorylation

Question 9

What is G₀ in the cell cycle?

Answer 9

The quiescent or waiting state

This is when the cell has not received enough mitogenic signalling to enter into G₁

Question 10

p53: what is it and what does it do?

Answer 10

Tumour suppressor that polices the cell cycle

• p53 checks genome stability before entrance into the S phase
• p53 also checks genome damage throughout the S phase
• p53 then prevents entry into the final cytokinesis phase if DNA is not fully replicated

Question 11

pRB: what is it, what does it do, and how is it regulated?

Answer 11

Tumour suppressor that polices the cell cycle

Acts as the restriction point - the point before growth inhibitors affect halting the cell cycle process: after this point, the cell cycle will be undergone regardless of external stimuli (internal concerns like DNA damage may still stop the process, however)

CDK4/6 phosphorylates pRB which is then hyperphosphorylated by B-CDK2

Question 12

Cyclin-CDK complexes: what are they, what do they do, how does their (de)activation work, and what are some specific examples?

Answer 12

Ser/Thr protein kinases that bind cyclins through a conserved alpha-helical motif PSTAIRE

Catalyse transitions in the cell cycle using the cyclin partner for substrate recognition and catalytic activity

Activation (T-) loop phosphorylation, rapidly degraded by the proteasome, ensuring one-directional movement through the cell cycle

• D-CDK4/6 drives cells to the R point
• E-CDK2 drives cells from G₁ to the S phase
• A-CDK2 drives cells through the first part of the S phase
• A-CDC2 drives cells through the S phase into the G₂ phase
• B-CDC2 drives the cell through mitosis

Question 13

Numerous small molecule and antibody therapies targeting mitogenic signalling and the cell cycle have been developed and tested

Answer 13

Mitogenic signalling repressors:
* Anti-Erb8 (Herceptin, Abx, etc) monoclonal antibodies (MoAbs)
* Human epidermic growth factor receptor (HER) 1/2/3 inhibitors (OSI-774, GW-2026, etc)
* Ras farnesyltransferase inhibitors (BMS-214662)
* RAF inhibitors (BAY 43-9006)
* MEK inhibitors (Cl-1040)
* mTOR inhibitors (RAD001)

Small-molecule cdk inhibitors may be classified based on effects on the cell cycle cdks:
* Pan-cdk inhibitors, including flavopiridol and AG (inhibit CDK4/6,2,1)
* Selective CDK inhibitors: 4/6 - PD,CINK4. 2,1 - Seliciclib, AZ703, etc