PATHOLOGY · PART I

Molecular Basis
of Cancer – I

Cell cycle · Signalling · Oncogenes

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SESSION OBJECTIVES

Three linked objectives for today

PA 7.2.1

Describe the cell cycle

PA 7.2.2

Write a note on cell signalling pathways

PA 7.2.3

Describe the role of proto-oncogenes, oncogenes and oncoproteins in carcinogenesis

Aim

Link each topic to the growth of a cancer cell

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CLINICAL HOOK

A 35-year-old man with a huge spleen

1Fatigue for some months
2Massive splenomegaly on examination
3Marked leucocytosis on blood counts
4Karyotype: t(9;22)(q34;q11)
5Which gene has been switched ON – and how?

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ROADMAP

From normal growth control to its hijack

1 · Cell cycle

How a cell divides – phases, cyclins, checkpoints

2 · Signalling

How the message to divide travels from surface to nucleus

3 · Oncogenes

How mutated growth genes drive cancer

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01

The cell cycle

How a cell decides to divide

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01 · CELL CYCLE

What is the cell cycle?

1Cell cycle: the period between two successive cell divisions
2Four unequal phases: G1, S, G2 and M
3Interphase = G1 + S + G2 – the time between mitoses
4G0: quiescent, resting cells outside the cycle

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01 · CELL CYCLE

Interphase: grow, copy, check

1G1: the cell grows in mass; the centrosome duplicates
2S: nuclear DNA is synthesised – each chromosome now has two sister chromatids
3G2: the G2/M checkpoint checks the copied DNA
4Cyclin B–CDK1 then drives the cell from G2 into M

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01 · CELL CYCLE

M phase: mitosis, then cytokinesis

1Prophase: chromosomes condense; nuclear envelope breaks down
2Metaphase: chromosomes line up at the equator
3Anaphase: sister chromatids separate to opposite poles
4Telophase: nuclear membranes re-form
5Cytokinesis: the cytoplasm divides – two daughter cells

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01 · CELL CYCLE

G1 sets the pace; G0 is the resting state

1The length of G1 separates slow from rapid dividers
2Some cells cycle every 16–24 hours; others never divide
3Restriction point (late G1): once past it, the cell is committed to divide
4G0: quiescent cells can re-enter G1 when stimulated

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01 · CELL CYCLE

The whole cycle at a glance

1Outer ring: G1 → S → G2 → M
2M: prophase, metaphase, anaphase, telophase
3Inner ring: the cyclin–CDK pair at each step
4After M: re-enter G1 or exit to G0

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01 · CELL CYCLE

Labile, stable and permanent cells

1Labile (go): divide throughout life – skin, gut, airway, marrow
2Stable (ready to go): rest in G0 after adolescence
3Stable cells divide when stimulated – e.g. liver after injury
4Permanent (stop): neurons, cardiac and skeletal muscle never divide

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01 · CELL CYCLE

Cell types compared

Feature	Labile	Stable	Permanent
Traffic signal	Green – go	Yellow – ready to go	Red – stop
Ability to divide	Throughout life	Falls after adolescence	Lost around birth
Cell-cycle status	Cycle mitosis to mitosis	Rest in G0; can re-enter	Have left the cycle
Examples	Epidermis, mucosa, marrow	Liver, kidney, fibroblasts	Neurons, cardiac muscle
After injury	Regenerate readily	Regenerate on stimulus	Die; no regeneration

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Walk across each row. Regeneration depends upon the dividing ability of the parenchymal cells. Traffic-light analogy: green means go – labile cells keep dividing throughout life (surface epithelium of skin, alimentary, respiratory and urinary tracts, vagina, cervix, endometrium, and haematopoietic cells of marrow, lymph nodes and spleen). Amber means ready to go – stable cells decrease their proliferation after adolescence but rest in G0 and can be stimulated to re-enter the cycle (liver, pancreas, kidney, adrenal, thyroid; mesenchymal cells such as smooth muscle, fibroblasts, endothelium, bone and cartilage). Hepatocytes are the classic example. Red means stop – permanent cells lose the ability to divide around birth (neurons, skeletal and cardiac muscle) and die after injury. Regeneration depends on the dividing ability of the parenchymal cells.

01 · CELL CYCLE

Cyclins rise and fall; CDKs wait for them

1Cyclins are made and destroyed in cycles – D, E, A, B in turn
2CDKs are always present but active only when bound to a cyclin
3Cyclin degraded → CDK activity wanes
4CDK inhibitors dock and switch the complex off

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01 · CELL CYCLE

Cyclin–CDK pairs through the cycle

Cyclin	Partner CDK	Acts at	Key job
Cyclin D	CDK4, CDK6	G1 → S	Phosphorylates Rb
Cyclin E	CDK2	G1 → S	Phosphorylates Rb
Cyclin A	CDK2, CDK1	S phase	Active during DNA synthesis
Cyclin B	CDK1	G2 → M	Essential for entry into mitosis

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01 · CELL CYCLE

CDK inhibitors put on the brakes

1Cyclin–CDK complexes phosphorylate targets such as Rb
2INK4 (p15, p16, p18, p19): block cyclin D–CDK4/6
3CIP/KIP (p21, p27, p57): block all cyclin–CDKs
4p53 induces p21; TGF-β induces p27
5Defective CDKIs: cells with damaged DNA divide

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01 · CELL CYCLE

Rb holds E2F – until phosphates land

1Active (hypophosphorylated) Rb binds E2F and holds the cell in G1
2Cyclin D–CDK4/6 adds phosphate groups to Rb
3Cyclin E–CDK2 adds more; Rb lets go
4Free E2F switches on S-phase genes
5Restriction point passed: no further growth signal needed

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01 · CELL CYCLE

Checkpoints close the gate on damaged DNA

1Checkpoints: surveillance that stops cells with genetic errors
2G1/S: checks DNA integrity before replication
3Fault found → cycle delayed, DNA repair triggered
4G2/M checks the copy; the spindle checkpoint* holds metaphase
5Irreparable damage → apoptosis or senescence (mainly p53)

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01 · CELL CYCLE

G1 → S: the main target in cancer

Cyclin D

Translocation → mantle cell lymphoma

Cyclin E

Overexpression → breast cancer

CDK4

Amplification → melanoma, glioblastoma, sarcomas

Defective CDKIs

Cells with damaged DNA keep dividing

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RECAP · PART 1

Cell cycle in four lines

Phases

G1 → S → G2 → M; G0 rests

Engines

Cyclin–CDK pairs, one per step

Brakes

CDKIs, Rb–E2F, checkpoints

In cancer

G1 → S control is broken

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02

Cell signalling

How the message to divide arrives

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02 · SIGNALLING

Cells live by the signals they receive

1Cells receive a constant stream of signals
2Signals direct growth, differentiation and function
3Sources: neighbouring cells, ECM (integrins), growth factors, cytokines, hormones
4No survival signal → the cell dies by apoptosis
5Lost "social controls" → unregulated growth (cancer)

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02 · SIGNALLING

Paracrine and autocrine signals

1Paracrine: acts on cells in the immediate vicinity
2Most growth factors act this way
3Autocrine: the secreting cell responds to its own signal
4Cancer cells may make – and respond to – their own growth factor

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02 · SIGNALLING

Endocrine and synaptic signals

1Endocrine: hormone released into the blood
2Carried to distant target cells
3Synaptic: neurotransmitter crosses a synapse to its target

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02 · SIGNALLING

From ligand to response: the basic sequence

1Ligand binds its receptor – high affinity, exquisite specificity
2Receptor clusters or changes shape
3Kinases add phosphates (phosphatases remove them)
4Transcription factors enter the nucleus
5Gene expression changes → cell response

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02 · SIGNALLING

Five receptor families carry the signal

1RTK: kinase tail – receptors for EGF, PDGF, insulin
2Non-receptor TK: a separate kinase binds (Src, ABL, JAK2)
3GPCR: 7-pass; G protein swaps GDP for GTP → cAMP, IP3
4Nuclear receptors: lipid-soluble ligands (steroids, vitamin D)
5Notch, Wnt/β-catenin: signal pieces move to the nucleus

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Receptor tyrosine kinases (RTKs): integral membrane proteins with a kinase tail, e.g. receptors for insulin, EGF and PDGF; ligand causes dimerisation and autophosphorylation of tyrosine residues; adaptor proteins couple the receptor to RAS, and it also recruits PI3K. Most oncogenic growth factor receptors belong to this class. Non-receptor tyrosine kinases: the receptor itself has no catalytic activity (e.g. immune receptors, some cytokine receptors, integrins); a separate cytoplasmic kinase binds after ligand binding. Src, the cellular homologue of the transforming protein of Rous sarcoma virus, is the prototype; SH2 domains bind phosphorylated receptors and SH3 domains mediate protein–protein binding. ABL (in CML) and JAK2 (in myeloproliferative disorders) belong here. GPCRs: seven-transmembrane (serpentine) receptors, more than 1500 identified; ligand binding makes the G protein swap GDP for GTP; second messengers cAMP and IP3; IP3 releases calcium from the endoplasmic reticulum. Nuclear receptors: lipid-soluble ligands (steroids, vitamin D) enter the cell and the complex binds DNA. Notch: ligand on an adjacent cell; the receptor is cleaved and intracellular Notch enters the nucleus. Wnt/β-catenin: Wnt binds Frizzled, the degradation complex is disrupted, β-catenin is stabilised and enters the nucleus (Wnt matters in colon carcinoma). GPCR and tyrosine-kinase receptors mainly drive proliferation.

02 · SIGNALLING

A growth factor switches on its RTK

1Growth factor binds the receptor tyrosine kinase
2Two receptors pair up (dimerise)
3Tyrosines on the tails are autophosphorylated
4Adaptor proteins bind the phosphotyrosines
5Adaptor couples the receptor to RAS; PI3K also docks

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02 · SIGNALLING

RAS–MAPK: the relay to the nucleus

1Inactive RAS holds GDP
2Signal: GDP replaced by GTP → RAS ON
3RAS activates RAF → MEK → ERK (MAP kinases)
4In the nucleus, transcription factors (e.g. MYC) switch on growth genes
5GAP boosts GTPase: GTP → GDP, signal OFF

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Binding of growth factor causes RTK dimerisation and autophosphorylation. Adaptor proteins couple the receptor to inactive GDP-bound RAS; GDP is displaced by GTP, giving active RAS. Active RAS activates RAF (a MAP kinase kinase kinase), which leads to phosphorylation of MAP kinases; these phosphorylate cytoplasmic proteins and nuclear transcription factors such as MYC, leading to transcription of growth genes and cell-cycle progression. MYC is an immediate early gene induced by RAS/MAPK signalling. The relay is switched off when RAS hydrolyses its GTP back to GDP: RAS has its own GTPase activity, accelerated more than 1000-fold by GTPase-activating proteins (GAPs). For every phosphorylation event there is a phosphatase that removes phosphate and usually inhibits signalling. Normal signalling is transient and limited. Standard teaching point (not in the source text): the source text names RAF (a MAP kinase kinase kinase) and MAP kinases; MEK (the MAP kinase kinase) and ERK (the MAP kinase) are the usual names of the middle and last kinases.

02 · SIGNALLING

PI3K–AKT–mTOR, with PTEN as the brake

1Phosphorylated RTK also recruits PI3K
2PI3K activates AKT – a node with >150 targets
3AKT → mTOR: more protein synthesis
4AKT blocks apoptosis (BAD, FOXO)
5PTEN, a tumour suppressor, restrains PI3K

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02 · SIGNALLING

Every step can go wrong in cancer

Growth factor

Overproduced – autocrine loop

➜

Receptor

Active without its ligand

➜

RAS

Locked GTP-bound "on"

➜

Kinase

Fusion always on (BCR–ABL)

➜

Nucleus

MYC, cyclin D/CDK4 overactive

Genes at every step are proto-oncogenes

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RECAP · PART 2

Signalling in four lines

Types

Paracrine, autocrine, endocrine, synaptic

Receptors

RTK, non-receptor TK, GPCR, nuclear, Notch/Wnt

Main relay

RTK → RAS → RAF → MAPK → nucleus

Second arm

PI3K → AKT → mTOR; PTEN brakes

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03

Oncogenes

When growth genes go rogue

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03 · ONCOGENES

Proto-oncogene, oncogene, oncoprotein

1Proto-oncogene: a normal growth-promoting gene
2Oncogene: its mutated or overexpressed form
3Oncoprotein: the protein an oncogene encodes – constitutively active
4First found in viruses (v-onc); about 100 described

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03 · ONCOGENES

Stuck accelerator vs failed brake

1Oncogene: gain of function – accelerator stuck on
2Dominant: one mutant allele is enough
3Tumour-suppressor gene: loss of function – brakes removed
4Recessive: usually both alleles lost (next session)

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03 · ONCOGENES

Oncogene vs tumour-suppressor gene (preview)

Feature	Oncogene	Tumour-suppressor gene
Derived from	Mutated proto-oncogene	Mutated growth-suppressor gene
Type of change	Gain of function	Loss of function
Alleles affected	One (dominant)	Usually both (recessive)
Genetic lesion	Point mutation, translocation, amplification	Deletion, point mutation, chromosome loss
Net effect	Accelerator stuck on	Brakes removed
Examples	RAS, MYC, ABL, HER2	RB, p53, APC

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03 · ONCOGENES

Four ways a proto-oncogene is activated

Mechanism	What happens	Classic examples
Point mutation	Single base change → protein always active	RAS, BRAF, c-KIT, RET
Translocation	Gene moves to a new site, escapes control	BCR–ABL (CML); MYC t(8;14) (Burkitt); cyclin D1 (mantle cell)
Amplification	Extra gene copies → overexpression	HER2 (breast); N-MYC (neuroblastoma); CDK4
Overexpression	More of a normal protein	SIS/PDGF-β (gliomas, sarcomas); EGFR

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03 · ONCOGENES

Mutant RAS cannot switch itself off

1Normal: GAP-boosted GTPase turns RAS off
2Point mutation: a single base change in RAS
3GTPase markedly reduced → RAS stuck GTP-bound
4Nonstop pro-growth signal: cells pile up
5RAS: commonest oncogene abnormality; loss of NF1 (a GAP) mimics it

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A point mutation is an alteration of a single base in the DNA chain. RAS proteins are small G proteins that flip between a signal-transmitting GTP-bound "on" state and a quiescent GDP-bound "off" state. A growth signal replaces GDP with GTP; activation is transient because RAS GTPase activity, accelerated by GAPs, hydrolyses GTP and switches RAS off. Point mutations markedly reduce GTPase activity, so RAS stays GTP-bound and the cell receives pro-growth signals continuously. Loss of the GAP neurofibromin (NF1) mimics this. RAS is the most common oncogene abnormality in human tumours – one source says about a third of tumours, another 15–20% overall with much higher rates in pancreatic adenocarcinoma; quote the general idea rather than a single figure. KRAS: colon, lung and pancreatic tumours; HRAS: bladder and kidney tumours. Other point-mutated oncogenes: c-KIT in GIST, RET in MEN 2A/2B and medullary carcinoma thyroid, FLT3 in AML, BRAF in melanomas and hairy cell leukaemia. Drugs to inhibit RAS have been difficult, because restoring a missing enzyme activity is hard.

03 · ONCOGENES

Important oncogenes and their tumours

Oncogene	Activation	Associated tumours
KRAS	Point mutation	Colon, lung, pancreatic tumours
BRAF	Point mutation	Melanoma, hairy cell leukaemia
ABL (BCR–ABL)	Translocation	CML, some acute leukaemias
JAK2	Point mutation	Myeloproliferative disorders
MYC	Translocation	Burkitt lymphoma
N-MYC	Amplification	Neuroblastoma
Cyclin D1	Translocation	Mantle cell lymphoma
CDK4	Amplification	Glioblastoma, melanoma, sarcoma

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Most oncogenes encode components of the cell proliferation signalling system, so they fall into five groups that follow the path of a growth signal: (1) growth factors – PDGF-β, TGF-α, FGF, HGF; (2) growth factor receptors – EGFR, HER2, c-KIT, RET; (3) cytoplasmic signal transducers – RAS, ABL–BCR; (4) nuclear transcription factors – the MYC family; (5) cell-cycle regulators – cyclin D, CDK4. Walk across the figure from the cell surface to the nucleus. Growth factors normally act in a paracrine fashion; a cancer cell may make and respond to its own growth factor (autocrine), e.g. glioblastomas expressing PDGF and PDGF receptor. ERBB1 (EGFR) mutations occur in a subset of lung adenocarcinomas. MYC, the master transcription factor on chromosome 8q, turns on growth genes including D cyclins and boosts ribosome production and glycolysis (Warburg effect); c-MYC is translocated in Burkitt lymphoma, N-MYC amplified in neuroblastoma, L-MYC amplified in small cell carcinoma lung. Learn one example per mechanism: point mutation – RAS; translocation – BCR–ABL and MYC; amplification – HER2 and N-MYC.

03 · ONCOGENES

t(9;22): two chromosomes break and swap

1ABL sits on 9q34; BCR on 22q11
2Both chromosomes break
3The pieces exchange – a reciprocal translocation
4Short chromosome 22 = Philadelphia (Ph) chromosome
5BCR–ABL hybrid gene → chimeric fusion protein

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03 · ONCOGENES

BCR–ABL: a kinase that never stops

1BCR–ABL: chimeric protein with tyrosine kinase activity
2BCR self-association unleashes ABL
3Kinase fires constantly → growth signalling never stops
4Kinase inhibitors block it: "oncogene addiction"
5CML stem cells persist – therapy must continue

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03 · ONCOGENES

Amplification: one gene, many copies

1Amplification: extra copies of a proto-oncogene
2More copies → the oncoprotein is overexpressed
3HER2 (ERBB2): breast, ovary, stomach – receptors crowd the cell surface
4N-MYC: neuroblastoma – double minutes or HSR
5HER2-amplified tumours respond to drugs that block HER2

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03 · ONCOGENES

MYC switches on many growth genes

1MYC: nuclear transcription factor (chromosome 8q)
2Immediate early gene, induced by RAS/MAPK
3Enters the nucleus; switches on growth genes, including D cyclins
4Boosts ribosome production and glycolysis
5c-MYC t(8;14) Burkitt · N-MYC neuroblastoma · L-MYC small cell lung

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03 · ONCOGENES

Five classes of oncogenes follow the signal

1 · Growth factors2 · Receptors3 · Signal transducers4 · Transcription factors5 · Cell-cycle regulators

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Most oncogenes encode components of the cell proliferation signalling system, so they fall into five groups that follow the path of a growth signal: (1) growth factors – PDGF-β, TGF-α, FGF, HGF; (2) growth factor receptors – EGFR, HER2, c-KIT, RET; (3) cytoplasmic signal transducers – RAS, ABL–BCR; (4) nuclear transcription factors – the MYC family; (5) cell-cycle regulators – cyclin D, CDK4. Walk across the figure from the cell surface to the nucleus. Growth factors normally act in a paracrine fashion; a cancer cell may make and respond to its own growth factor (autocrine), e.g. glioblastomas expressing PDGF and PDGF receptor. ERBB1 (EGFR) mutations occur in a subset of lung adenocarcinomas. MYC, the master transcription factor on chromosome 8q, turns on growth genes including D cyclins and boosts ribosome production and glycolysis (Warburg effect); c-MYC is translocated in Burkitt lymphoma, N-MYC amplified in neuroblastoma, L-MYC amplified in small cell carcinoma lung. Learn one example per mechanism: point mutation – RAS; translocation – BCR–ABL and MYC; amplification – HER2 and N-MYC.

03 · ONCOGENES

Growth factors and receptors as oncogenes

Oncogene	Encodes	Activation	Tumours
SIS	PDGF-β	Overexpression	Gliomas, sarcomas
HST-1, INT-2	FGF	Overexpression, amplification	Bowel, breast cancer
ERBB1	EGFR (HER1)	Overexpression, mutation	SCC lung, glioblastoma
ERBB2	HER2/neu	Amplification	Breast, ovary, stomach
c-KIT	Stem cell factor receptor	Point mutation	GIST
RET	RET receptor	Point mutation	MEN 2A/2B, medullary Ca thyroid

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03 · ONCOGENES

Case closed: BCR–ABL drives his CML

1t(9;22) = the Philadelphia chromosome
2BCR–ABL fusion defines CML – the initiating event
3Kinase inhibitors work by "oncogene addiction"
4Our patient: BCR–ABL-driven CML

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HIGH-YIELD · I

Cell cycle and signalling: must-know

1Cell cycle: G1, S, G2, M; G0 is the resting state
2Length of G1 separates slow and rapid dividers
3Labile (go), stable (ready to go), permanent (stop) cells
4Cyclin D–CDK4/6 and cyclin E–CDK2 phosphorylate Rb at G1/S
5CDKIs: CIP/KIP (p21, p27, p57) and INK4 (p15, p16, p18, p19)
6Signalling: paracrine, autocrine, endocrine, synaptic
7RTK → RAS → RAF → MAPK; and PI3K → AKT → mTOR

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HIGH-YIELD · II

Oncogenes: must-know

1Proto-oncogene (normal) → oncogene (mutant) → oncoprotein
2Oncogenes are dominant: one mutant allele is enough
3Mechanisms: point mutation, translocation, amplification
4RAS: commonest; point mutation locks it in the GTP state
5BCR–ABL, t(9;22): constant tyrosine kinase in CML
6MYC: t(8;14) Burkitt; N-MYC amplified in neuroblastoma
7HER2 (ERBB2) amplified in breast cancer

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SUMMARY

One map: growth control and its hijack

Cell cyclePhases · cyclins · checkpoints

➜

SignallingReceptor → RAS–MAPK / PI3K–AKT → nucleus

➜

OncogenesAccelerator stuck ON

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Thank you

Questions and discussion

Next session: tumour-suppressor genes, DNA repair and the other hallmarks

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Molecular Basisof Cancer – I

Three linked objectives for today

A 35-year-old man with a huge spleen

From normal growth control to its hijack

What is the cell cycle?

Interphase: grow, copy, check

M phase: mitosis, then cytokinesis

G1 sets the pace; G0 is the resting state

The whole cycle at a glance

Labile, stable and permanent cells

Cell types compared

Cyclins rise and fall; CDKs wait for them

Cyclin–CDK pairs through the cycle

CDK inhibitors put on the brakes

Rb holds E2F – until phosphates land

Checkpoints close the gate on damaged DNA

G1 → S: the main target in cancer

Cell cycle in four lines

Cells live by the signals they receive

Paracrine and autocrine signals

Endocrine and synaptic signals

From ligand to response: the basic sequence

Five receptor families carry the signal

A growth factor switches on its RTK

RAS–MAPK: the relay to the nucleus

PI3K–AKT–mTOR, with PTEN as the brake

Every step can go wrong in cancer

Signalling in four lines

Proto-oncogene, oncogene, oncoprotein

Stuck accelerator vs failed brake

Oncogene vs tumour-suppressor gene (preview)

Four ways a proto-oncogene is activated

Mutant RAS cannot switch itself off

Important oncogenes and their tumours

t(9;22): two chromosomes break and swap

BCR–ABL: a kinase that never stops

Amplification: one gene, many copies

MYC switches on many growth genes

Five classes of oncogenes follow the signal

Growth factors and receptors as oncogenes

Case closed: BCR–ABL drives his CML

Cell cycle and signalling: must-know

Oncogenes: must-know

One map: growth control and its hijack

Thank you

Molecular Basis
of Cancer – I