Lecture 1: The Metastatic Process

• General Overview and Mechanisms

• Pre-existent subpopulations

• Lymphangiogenesis

• Specificity of metastatic patterns of spread

Differences between Benign and Malignant Neoplasms

BENIGN MALIGNANT 1.Often encapsulated Non encapsulated 2.Well differentiated Poorly differentiated 3.Low mitotic rate High mitotic rate 4.Noninvasive Invasive 5.Non-metastasising Metastasising

Benign tumours are characterised by:

• Localised • Usually grow slowly • Compress, rather than infiltrate, adjacent

tissue, often forming a capsule • Do not spread to distant sites generally do

not recur after removal • May be self limiting or regress • Have cellular structure resembling that of

the tissue of origin

Malignant tumours are characterised by:

• Infiltration of malignant cells into surrounding tissues

• Invasion of neoplastic cells into blood and lymph vessels

• Spread of tumour cells to other parts of the body to establish secondary growths (metastasis)

METASTASIS

The transfer of disease from one

organ

or part of an organ to another not

directly connected with it

Spread of Cancer

• No invasion – in situ

• Local invasion • Dissemination

– Lymphatics – Blood stream – Intracavitary – Perineural

How Cancer Kills ?

• Late presentation is the norm – 109 cells per cubic cm of cancer

• Smallest lump detectable is about 1cm

• Local effects if anatomically problematic

- blocks tubes

- bleeds

- obstructs glands

• Metastatic burden = 1kg = death

Metastasis: the spread of tumours

Primary tumour

Secondary tumour

Intravasation

Extravasation

B16 Selection - 1973

F1

F10

Does it represent selection or adaptation?

Interpretation

• Metastatic sub-populations pre-exist within the parental population.

• These sub-populations breed true therefore there must be a genetic basis to their metastatic capacity.

• Added later- Gene expression signatures between primary and metastatic tumours should be different.

a

b c

Interpretation

Gene expression signatures of primary and secondary tumours

are similar.

Conclusions • Metastasis probably is regulated by genes we

already know about.

• How those genes are regulated though is not necessarily obvious.

• Technological developments ( gene expression arrays, miRNA arrays ) will allow us to interrogate this process.

• In future the focus may need to be on how disseminated cells grow at distant sites rather than how they accomplish this process.

Role of Angiogenesis in Tumour Spread

ANGIOGENESIS and METASTASIS

Weidner et al (1992) Prognostic Factor in Breast Cancer Horak et al (1992) Correlation with Survival in Breast Cancer MacChiarini et al (1992) Correlation with Metastasis in Lung Cancer Weidner et al (1993) Correlation with Metastasis in Prostate

Cancer

Angiogenic peptides

Angiogenic peptides

SS SS

NRP-1 NRP-1

VEGFR-1 VEGFR-2 VEGFR-3

VEGF-A

VEGF-B

VEGF-C

VEGF-D OV-VEGF

PIGF

VEGF-B

sVEGFR-1

P1114L

P P P P P P

Migration, permeability, DNA synthesis, survival

angiogenesis lymphangiogenesis

LYMPHANGIOGENESIS

• VEGF-C/VEGF-D binding to VEGF-R3.

• LYVE-1 as a marker of lymph vessels.

• Experimentally VEGF-C has promoted breast cancer metastasis.

• Clinical correlations with metastatic activity e.g. gastric,cervical and breast

LYMPHANGIOGENESIS

• Are the identified lymphatics functional?

• How specific is LYVE-1 as a marker?

• How solid are the clinical data?

• Is there variation between tumour types?

Metastasis: the spread of tumours

Primary tumour

Secondary tumour

Intravasation

Extravasation

“Cancer invasion can be viewed as a derangement in the proper sorting of cell populations, causing a violation of

normal tissue boundaries”

Liotta and Kohn

Nature 411: 375-379, 2001

Cellular Invasion: 3-step Process

• 1. Cell Attachment

• 2. Degradation of ECM

• 3 Cell Migration

Proteinases Serine proteinases uPA, thrombin,

Cathepsin G Cysteine proteinases Cathepsin B, L. Aspartyl proteinases Cathepsin D Matrix metalloproteinases Gelatinases,

Collagenases

SERINE PROTEASES

• Serine residue at active site

• Inactive pro-form

• uPA, trypsin, plasmin

Urokinase type Plasminogen activator (uPA)

• Plasminogen plasmin

• Concentrated at cell surface

• Tumour-stromal interface

MMPs • family of related enzymes with shared

domains • degrade collagens and other matrix proteins • Zinc dependent enzymes, work best at neutral

pH • generally secreted as inactive pro-enzymes • all inhibited by “TIMPs”

M2+ D S

P G R

G

Pax Tensin

FAK src G

M2+ D S

P G R

G

Pax Tensin

FAK G

a-actinin

actin

ECM glycoprotein

eg fibronectin

Cation-binding

site

SS SS

Small GTPases

-Rho,Rac,cdc42

a b

Selectins Integrins

Rolling Activation Flattening and

firm adhesion

Transendothelial

migration

CHEMOATTRACTANT (eg released after injury or infection)

Leukocyte Extravasation

The Nonrandom Nature of Metastatic

Spread

1.Hemodynamic Factors Increased bloodflow = increased frequency 2. Specific Cell Recognition Cell receptors = increased trapping 3.Seed and Soil Tumour/organ growth interaction = metastasis

Figure 1 The hypoxic response. a, Under conditions of normal oxygen, the von Hippel–Lindau tumour suppressor protein

(pVHL) modifies the protein HIF, which leads to its destruction. b, When oxygen is scarce (hypoxia), or when pVHL is

mutated, HIF accumulates inside cells and activates the expression of certain genes. This triggers two complementary

responses. First, tissue oxygenation is stimulated through the activation of genes such as VEGF (which stimulates the

outgrowth of new blood vessels) and erythropoietin (EPO, which stimulates the production of red blood cells). Second,

tumour cells are stimulated to move away from the site of hypoxia through the activation of genes such as c-Met, which

enhance cell motility and invasion. Now, Staller et al.3 show that the gene CXCR4 is also activated by HIF. CXCR4 not only

stimulates migration, it also enables tumour cells to

Cancer metastasis and chemokine signalling. Initiated epithelial cells are promoted by inflammation to undergo neoplastic progression, a process

that requires remodelling of the extracellular matrix, recruitment of inflammatory cells, angiogenesis and lymphangiogenesis. Out of this

microenvironment, carcinomas arise. These neoplastic cells then turn on expression of chemokine receptors, such as CXCR4. The production of

chemokine ligands for these receptors, in sites such as lymph nodes, bone marrow, liver and lung, then facilitates their invasion and migration to

secondary sites where malignant cells reside either in a dormant state, or proliferate to form a productive metastatic lesion. Blockade of

chemokine receptors, for example, anti-CXCR4 antibodies, attenuates metastatic spread in some experimental systems.

Metastasis: the spread of tumours

Primary tumour

Secondary tumour

Intravasation

Extravasation

Lecture 2: Intercellular adhesion and metastasis

• Cadherin superfamily

• Selectin family

• Integrin family

A functional classification of cell junctions

1. Occluding junctions (tight junctions)

2. Anchoring junctions

a. Actin filament attachment sites (adherens junctions)

i. cell-cell (e.g. adhesion belts)

ii cell-matrix (e.g. focal contacts)

b. Intermediate filament attachment sites

i cell-cell (desmosomes)

ii cell-matrix (hemidesmosomes)

3. Communicating junctions

a. gap junctions

b. chemical synapses

c. plasmodesmata (plants only)*

Tight Junction

(Zonula Occludens)

Lumen

Gap

Junction Hemidesmosome

Belt Desmosome

(Zonula Adherens)

Basement

Membrane

Extracellular Matrix

Cadherin Superfamily

Subfamily Members

Classical cadherins E-,N- or P-Cadherin

VE-Cadherin

Desmosomal cadherins Desmocollin

Desmoglein

Proto-cadherins -Protocadherin

CNR-Cadherin

Seven transmembrane (7TM) Flamingo

cadherins

T-cadherin T-cadherin

FAT family Dachsous

Fat

G protein

β-cat

DP

PG

PP

FYN

PDZ

β-cat α-cat

β-cat α-cat

vinc

PG

vinc

DP

DP

vinc

actin

IF

actin

IF

IF

mDAB1

Signalling cascades

eg. SRC protein tyrosine

kinases, G-proteins

?

E-, N- or

P-cadherin

VE-cadherin

classical

cadherins

desmocollin

desmoglein

μ-protocadherin

CNR-cadherin

protocadherins

7TM-cadherin

T-cadherin

FAT-family cadherin

Calcium dependent, cell-cell adhesion molecules Numerous family members, cell-lineage specificity of expression e.g. E-cadherin in epithelial cells Extracellular domain has 5 homologous repeats Binding specificity located to most distal repeats Establish and maintain intracellular connections Down-regulation results in cell dispersal

CADHERINS

Produced as 135kDa glycoprotein After cytoplasmic trimming expressed as 120kDa molecule Five extracellular domains of – 110 amino acids each, internal sequence homology, conserved Ca2+ binding motifs

E-Cadherin

N

Pre-region

a

b C

E1 E2 E3 E4 E5

HAV

Catenin complex

Catenin Size Chromosomal location a 102kDa 5q31 b 92kDa 3p21 (plakoglobin) 83kDa 17q21 p120ctn (p120 cas) 11q11

p120ctn (p120cas)

P120cas – major Src (tyrosine kinase) substrate

Arm domain – 11 copies of a 42-amino-acid motif originally described for the Drosophila segment polarity gene product, armadillo

At least 4 different isoforms

Reduced p120ctn expression correlates with poor survival in

patients with adenocarcinoma of the gastroesophageal junction

Wijnhoven, BP et al J Surg Oncol 92:116-123, 2005.

Conversion of Epithelial to Mesenchymal

Downregulation of E-cadherin activity (RNAi, Antibody)

Restoration of E-cadherin (cDNA)

Table 1 ECD expression and Dukes stage

ECD expression A&B C1&C2 ECD++/ECD+ 32 7 ECD- 4 29 36 36 Fisher exact test p<<0.001

Table 2 ECD expression and tumour grade

ECD expression Well Moderate Poor ECD++ 7 4 0 ECD+ 1 24 4 ECD- 0 8 24

8 36 28

E-Cadherin Downregulation

• Transcriptional regulation e.g. increased „Snail‟ activity, promoter methylation.

• Mutation in E-cad gene and LOH.

• Catenin mutation and downregulation leads to loss of functional cadherin/catenin complex.

Concept of EMT

• Phenotypic switch

• Co-ordinated set of molecular changes

• Change in function,fate and character

Transient loss of basement memranes in malignant progression: Brabletz model.

Tumour microenvironment

ZEBI?

EMT + BM Loss

MET MET

Invasive front

dissemination

Taken from:- Spaderna et al

Gastroenterology 131: 830, 2006

Concept of EMT

• Phenotypic switch

• Co-ordinated set of molecular changes

• Change in function,fate and character

• Change in morphological appearance

Gene expression changes said to be associated with EMT

• Loss of- • E-cadherin expression • Cytokeratin expression

• Gain of- • Vimentin expression • N-cadherin expression • MMP expression

Human lobular and ductal breast carcinomas

lobular ductal

Transient loss of basement memranes in malignant progression: Brabletz model.

Tumour microenvironment

ZEBI?

EMT + BM Loss

MET MET

Invasive front

dissemination

Taken from:- Spaderna et al

Gastroenterology 131: 830, 2006

Conclusion

• Loss of E-cadherin associated with tumour progression and metastasis

Wnt1 receptor

1111111

Dsh

P

P

P

b a

b-catenin

APC

GSK-3b

b

LEF/TFC

Cyclin D1

myc

Nucleus

P GSK-3p

LEF/TCF

GSK-3b

b

APC

Proteasome

WNT-wingless Pathway

Wnt-1 protein extracellular signalling factor

Frizzled seven-transmembrane-domain receptor

for Wnt-1

Dishevilled (Dsh) phosphorylated to active form by

casein kinase II

Glycogen synthase kinase-3b complexes with APC and cytoplasmic b-catenin

(GSK-3b)

Adenomatous polyposis coli phosphorylated, together with b-catenin, by GSK-3b

(APC)

Phosphorylated b-catenin Ubiquitinated. Proteasome degradation

Association of Wnt-1 and Frizzled

results in activation of Wnt-1 signalling pathway

b-catenin transcription

LEF/TCF leucocyte enhancer factor/T-cell factor

Free b-catenin Translocates to nucleus

Plus LEF/TCF Induces DNA binding and transcription

c-MYC LEF/TCF-responsive genes

cyclin D1

Selectins

P-selectin platelet 140kDa

E-selectin endothelial 95-115kDa

L-selectin leucocytes 74kDa (lymphocyte)

90-100kDa (neutrophil)

N-terminal C-type lectin domain

Epidermal growth factor (EGF)-like motif

Short consensus repeats (variable) similar to those found in

complement-regulatory proteins

Recognise fucosylated carbohydrate ligands (especially sialyl-

Lewisx)

L-selectin

Constitutively expressed by leukocytes -majority B cells, virgin T cells, neutrophils, monocytes and eosinophils Mediates binding to activated endothelium at inflammatory sites Ligands are highly glycosylated mucin-like molecules Direct binding lymphocytes to HEV of peripheral lymph nodes

E-selectin Expressed by cytokine-activated endothelial cells

Protein synthesis-dependent

Peaks 4-6 hours post cytokine stimulation

Mediates neutrophil, monocyte, memory

T-cell adhesion

Can be found in serum (proteolytic cleavage from cell

surface). Elevated in various inflammatory syndromes

P-selectin

Constitutively expressed in Weibel-Palade bodies of endothelial cells and alpha granules of platelets Rapidly mobilised to surface in response to inflammatory agents Cell surface expression short-lived Mediates adhesion of neutrophils and monocytes to activated platelets and endothelial cells

Integrin Ligands

• Extracellular matrix components

(many contain RGD motif)

• Ig superfamily molecules (e.g. VCAM)

Additional Reading

Ramachandran V et al

Dimerization of a selectin and its ligand stablizes cell rolling and enhances tether strength in shear flow

Proc. Natl. Acad. Sci. USA 98: 10166-10171 2001

Michaelson JS and Leder P

b-catenin is a downstream effector of Wnt-mediated tumorigenesis in the mammary gland

Oncogene 20: 5093-5099 2001

Runswick SK et al

Desmosomal adhesion regulates epithelial morphogenesis and cell positioning

Nature Cell Biology 3: 823-830, 2001