Cellular Basis of Cancer 3

Proteases in CancerDr Rosemary Bass

rosemary.bass@northumbria.ac.uk

Outline

Protease = proteinaseWhy proteases are importantIntroduction to proteases:

Classes MMP (Matrix Metalloprotease)ADAM/ADAMTS (A Disintegrin and a Metalloprotease)Serine proteasesInhibitors (TIMPs, Serpins)

Mechanisms of actionHow enzymes are regulated – zymogens, spatial, temporal

Why proteases thought to promote cancer - Roles of extracellular proteases in tumour invasion & metastasis

Problems with broad-spectrum MMP inhibitors as anti-cancer therapy

Anti-cancer activities of proteases

Proteases – molecular scissors

- cleave proteins in vivo

- impact on many basic cell functions:

cell adhesion

cell division/ proliferation/multiplication

cell migration – invasion/metastasis

cell survival - apoptosis

Because alters cellular environment

•Proteases alter (tumour) cell environment•Cleavage of ECM

Cell are in contact with – other cells- extracellular matrix (ECM)

Extracellular Matrix = ECM

Outside cells

Secreted by cells

Consists of polymeric proteins eg. collagen in skin & bone

Collagens form 25% of total body protein

Glycoproteins eg. Fibronectin

Glycosoaminoglycans/proteoglycans eg. Aggrecan & hyaluronan in cartilage

Fibroblast surrounded by collagen fibres

Collagen fibres in cross-sectionCollagen fibres in longitudinal section

How do most often hear about collagen?

Initial interest in mechanical strength provided by ECM

cell adhesion

cell proliferation

cell migration

cell apoptosis

Interaction with ECM important for all major cell biological processes:

Proteases Cleave the ECM

IntegrinHeterodimer

-Cell surfaceadhesion receptors

S

S

M +

M +

M +

S

S

M+ M

+ M+

Inactive Active

•Connect cell to ECMActivation state can be regulated to control cell-ECM interactions

Activeectodomain Inactive llb3

51Predicted

Integrin Activation

Tissue RemodellingTissue Remodelling

NormalNormal PathologicalPathological

ReproductionReproductionOrganogenesisOrganogenesisWound repairWound repair

Tumour growthTumour growthMetastasisMetastasisArthritidesArthritidesAtherosclerosisAtherosclerosis

•Proteases participate in normal & pathological tissue remodelling

•In tissue remodelling proteases

•Normal tissue - tightly controlled, temporally regulated

There are 5 Catalytic Classes of Proteases

(Neutral pH, cell surface/extracellular)

(Intracellular, Important for cancer, caspases in apoptosis, proteasome targeting therapies)

1-Metalloproteinases: Matrix metalloproteinases (MMPs), disintegrin metalloproteinases (ADAMs and ADAMTSs)

2-Serine: plasmin, uPA, tPA, PMN elastase, proteinase 3, cathepsin G, kallikrein, tryptase, chymase, granzymes

3-Cysteine: Cathepsin B, L, S, K, calpains, caspases

4-Aspartate: Cathepsin D

5-Threonine: Proteasomal subunits

MMP MMP NumberNumber DOMAIN STRUCTUREDOMAIN STRUCTURE

Matrilysins MMP-7, -26

Collagenases MMP-1,-8,-13Metalloelastase MMP-12Stromelysins MMP-3,-10,-11Others MMP-19,-20,-27,-28

MMP-23 A/B

MT-MMPs MMP-14,-15,-16,

-17,-24,-25

Gelatinases MMP-2, -9

C Zn

PropeptideN-terminal domain

(catalytic)

C CCZn

C-terminal domain(hemopexin-like repeats)

C CCZn II

II

II

Gelatin binding domain(fibronectin-like repeats)

C CCZn

Membranedomain

C Zn

Cys array Ig-likeCys array Ig-like

•Classed according to domain structure•Catalytic domain & propeptide•All have Zn2+ binding site – target for inhibitors•C-term region involved in substrate binding & TIMP interactions•Membrane bound by transmembrane domain or GPI anchor

(23 in human, 24 in mouse)

MMP = Matrix Metalloprotease

Resorption of tail during tadpole→ frog metamorphosis – first identification of collagenase by Jerry Gross in 1965

MMP in normal development

Wound healing Epithelial migration Angiogenesis

ECM invasion by cancer cell

REGULATION OF METALLOPROTEINASES:

• Gene transcription

• Post-transcription

• Post-translation -pro forms require activation (plasmin, MMPs, furin/

Pro-Protein Convertases) -intracellular trafficking of membrane bound MPs -cell and ECM sequestration

-inhibition by endogenous TIMPs

Tissue Inhibitors of Metalloproteases (TIMPs)

TIMP-1TIMP-1

TIMP-2TIMP-2

TIMP-3TIMP-3

TIMP-4TIMP-4

SolubleSoluble

SolubleSoluble

ECM AssociatedECM Associated

SolubleSoluble

Leco Leco et al.,et al., (1994) J Biol Chem (1994) J Biol ChemLeco Leco et al., et al., (1997) FEBS Let(1997) FEBS LetBaker, Edwards & Murphy (2002) J Cell ScBaker, Edwards & Murphy (2002) J Cell Sc

Characteristic TIMP-1 TIMP-2 TIMP-3 TIMP-4

MW 28kDa 21kDa 24kDa 22kDa

Localization soluble soluble ECM soluble

Tissue specificity bone, ovary, lung, heart, kidney, heart, heart, (mouse) muscle, brain, brain, lung, muscle,

skin, vessels, ovary, brain, testis, ovary placenta ovary

Growth promotion yes yes no yes

Apoptosis no no yes yes

Inhibition of tumour + + + +invasion

Angiogenesis inhibition yes yes yes yes

Ability to inhibit no yes yes yes

MT1-MMP

MMP-TIMP BalanceMMP-TIMP Balance

MMP TIMP

MMP

Normal

ECM Homeostasis

TIMP

Disrupted Balance

Favours ECM Destruction

ADAMs – A Second Class of Metalloproteinase

AA DDisintegrin isintegrin aand nd MMetalloproteinaseetalloproteinase

Or AdamalysinOr Adamalysin

Involved primarily in “ectodomain shedding” Involved primarily in “ectodomain shedding” eg. TACE = ADAM17eg. TACE = ADAM17

Cytoplasmic tail of some ADAMs may be involved in inside–out Cytoplasmic tail of some ADAMs may be involved in inside–out control of proteinase activity or outside–in control of cell control of proteinase activity or outside–in control of cell signallingsignalling

Can be inhibited specifically by TIMP-3Can be inhibited specifically by TIMP-3

Seals & Courtneidge (2003) Genes and DevSeals & Courtneidge (2003) Genes and Dev

EGF

Cys-rich

Disintegrin

Metallo

The ADAM FamilyA Disintegrin and Metalloprotease

Snake venomMetalloproteinases

PP

Cytoplasmic

ADAM = A Disintegrin and A Metalloprotease

29 Mammalian ADAMs

Pro-domain – intramolecular chaperone inhibiting protease domainMetalloprotease domainDisintegrin domain = cell-cell adhesionCys-rich domain = cell-cell & cell-ECM domainEGF repeatsTransmembrane domainCytoplasmic tail

Spermatogenesis/fertilisationDe-regulation implicated in development of cancer

ADAMs and Cell Adhesion

A) Trans

B) Cis

Dis

EGF Dis

Integrin

Cys MP

Syndecans

•Involved in cell adhesion as well as proteolysis

•Signalling through multiple pathways

Shedding• Proteolytic cleavage of membrane-bound proteins (growth factors,

cytokines, receptors)• Post-translational modulation • Activate, inactivate or change properties of processed proteins

C Tape, CRI, Cambs

TNF convertase,TACE, ADAM-17

pro

MP

Disintegrin

Cys-richEGF-like

TM

Cyto

TNF

Proteins that are shed:

Cytokines, growth factorsTGF, EGF, HB-EGF, TNF, KL-1, CSF-1, FasL,Delta

ReceptorsTNFRI, TNFRII, p75 NGFRIL6R, TSHR

Adhesion proteins, othersL-selectin, syndecans, PTP, LAR , ACE, APP

Schlondorff J and Blobel C, (1999)J. Cell Sci. 112: 3603-3617

TACE – EGF signallingADAM 10 – notch, Ephrins

Differentially spliced isoforms of ADAM15 cytoplasmic tail in breast cancer

MCF

-7T4

7DBT-

549

MDA-M

B-231

ADAM

15a

CBAD

MX6

MX7

MX8

ADAM

15a

DC gr1 DC gr2 DC gr3 LC gr2

CBAD

Signal sequence

Prodomain

Metalloprotease

Disintegrin

Cysteine-rich

EGF-like

Transmembrane

Cytoplasmic tail

ADAM-15 Isoform

PMA - 1 3 7 24 48 72 96 ADAM

15a

BA

U937 cells PMA timecourse

Cell lines

Breast tissues and cancers

Zhong et al 2008 Mol Cancer Res 6:383

Levels of splice variants differs in breast cancer

Association with cell behaviour & agressiveness of cancer

ADAMTS = A Disintegrin and A Metalloprotease with Thrombospondin like motifs

19 secreted proteasesADAMTS1 – discovered 1997ECM degradation/assembyHaemostasisOrganogensisAngiogenesisDe-regulation associated with development arthritis & cancer

Signal peptide

Pro domain

Metalloproteinase

Disintigrin

Cysteine-rich

Hemopexin

TS repeat

Transmembrane Region

Spacer region

EGF like

Hinge

Cytoplasmic tail

Domain Key

x

ADAM SVMP ADAMTS MTMMP MMP

Serine Proteases

•plasmin, uPA, tPA, PMN elastase, proteinase 3, cathepsin G, kallikrein, tryptase, chymase, granzymes

•Catalytic triad

EGFFn3

Kr Kr Ser Pr

Ser PrKrEGF

uPA, urokinase plasminogen activator

tPA, tissue plasminogen activator

expressed by wide variety of cells virtually inactive zymogen binds to uPAR

very limited expression “active zymogen” activity stimulated by fibrin

KrKrKrKr Kr Ser PrNTPPlasminogen

synthesized in liver, most abundant protease zymogen very broad substrate specificity lysine-binding kringle modules

Cell

Extracellular Matrix(ECM)

Plasminogen Binding Sites

Plasminogen

uPAR

Pro-uPA

uPA

“tPAR”

tPA

PAI-1

PAI-1

Plasmin

2-antiplasmin

Activationpro-MMPs &Latent GrowthFactors

CD82

51

AdhesionMigrationSurvival

Proteases Facilitate Tumour Invasion & Metastasis

Normalcell

Transformedcell

BM

ECM

Bloodvessel

“Classic” idea that proteases are only acting like molecular scissors to promote invasion & metastasis

The main steps in metastasis

Since >80% of human tumours derive from epithelial cells (giving rise to carcinomas when malignant) we will mainly consider metastasis of these tumours, but essentially similar steps are involved in the spread of connective tissue tumours (sarcomas)

Epithelial cells sit on a basement membrane (basal lamina), with stroma = connective tissue underlying.Stroma includes fibroblasts, blood vessels, immune effector cells. Tumour-stromal interactions are of critical importance in metastasis

1990’s view of MMPs in Cancer Biology

•Multiple protease involvement•From different sources - cancer cells/macrophage/stromal cells•Still perceived that proteases all contributing to cancer progression•Therefore good targets for therapy protease expression in cancer vs normal tissue

Overlapping substrate preferences of MMPs

MMP Target

Fibroblast collagenase

Gelatinase-A

Gelatinase-B

Matrilysin

Stromelysin- 1

Collagen VII

Fibronectin

Laminin

Therefore it was thought to be a good idea to develop broad-spectrum MMP inhibitors that would block many/all family members

However broad-spectrum Matrix Metalloprotease Inhibitors (MMPI) have proved disappointing in the clinic

MMP1

Metalloproteasedomain

BB94(Batimastat)

MPI CANCER RESULT Marimastat x3 Pancreatic II-IV No benefitBB-2516 x1 Gastric (advanced ) Benefit after other treatment x1 Glioblastoma (unres) No benefit x1,x1Non-small/small cell lung No benefit x1 Ovarian (advanced) No benefit

Prinomastat x2 Non-small cell lung No benefit AG 3340 x1 Prostate (metastatic) No difference

Tanomastat x1 Small cell lung Poorer survival BAY 12-9566 x1 Pancreatic (metastatic) Ditto

BMS-275291 x1 Non small cell lung (IIIB,IV) Recruiting Neovastat x1 Renal cell carcinoma Recruiting

Coussens et al Science 2002

→Simple notion that all proteases are bad not correct

control batimastat AG3340 Ro28-2653 Ro206-0222

liver

met

asta

sis

[%]

0

50

100

200

250

The broad-spectrum MMPI batimastat The broad-spectrum MMPI batimastat increases increases metastasis in a murine L-CI.5s lymphoma model metastasis in a murine L-CI.5s lymphoma model

Arlt M. et al., Cancer Research, 2002.Increasing specificity for MMP9

THEN:

NOW:

PROTEASES IN CANCER

Coussens et al Science 2002

Tumour-stromal interaction is important for activating the protease cascade leading to MMP activation and ECM destruction

Tumours have elevated levels of many proteases, many of which are expressed by host stromal cells.

Proteases on the cell surface focus proteolysis on the cell surface

Proteases also regulated by location – where the activities are

Membrane-type-1 MMP activates pro-MMP-2Membrane-type-1 MMP activates pro-MMP-2(Gelatinase-A) on the cell surface(Gelatinase-A) on the cell surface

MT1-MMPMT1-MMP

TIMP-2TIMP-2

Pro-MMP-2Pro-MMP-2ActiveActiveMMP-2MMP-2

Good example of this is the requirement for TIMP-2 in theactivation of MMP2:

Fidler’s experiment with B16 melanoma cells suggested that there was a small metastatic sub-population of cells in a primary tumour that could be selected for

How do metastatic cells arise?

B16F0 = poorly metastaticB16F1 = low metastatic abilityB16F2,3,4,5,6,…………..B16F10 = highly metastatic

Fidler IJ 1973 Nature New Biology 242:148-149. Selection of successive tumour lines for metastasis

Experimental Metastasis Assay showing metastasis suppression effects of TIMP-1

B16F10 cells injected in control mice via tail vein

B16F10 cells injected along with recombinant TIMP-1 (and treated for 6.5days)

Metastasis assay showing lungs from mice injected with B16F10 tumour cells (highly metastatic melanoma cells)

Schultz et al., Cancer Res. 1988

Tumours

Clinical correlation of high expression of certain MMPs with poor outcome – 100s of studies

Eg. Colorectal cancer – poor survival with MMP9 expression

T=tumour, N=normal colon T N

MMP-9 equates with poor survival in colorectal cancer

Other key facts……

Kaplan-Myersurvivalcurve

So why did synthetic MMP inhibitors fail as anti-cancer agents?

• Trial design – MMPIs (in general) don’t work on very advanced cancers

• Inhibit not just MMPs but other metalloproteinases eg ADAMs whose existence was unknown

• MMPs can have unexpected anti-cancer roles

So why did synthetic MMP inhibitors fail as anti-cancer agents?

• Trial design – MMPIs (in general) don’t work on very advanced cancers

• Inhibit not just MMPs but other metalloproteinases eg ADAMs whose existence was unknown

• MMPs can have unexpected anti-cancer roles

So why did synthetic MMP inhibitors fail as anti-cancer agents?

• Trial design – MMPIs (in general) don’t work on very advanced cancers

• If animal model studies had been done properly would have found that MMPI don’t work on late stage cancers before clinical trails

Tumour progression in the RIP-Tag pancreatic cancer model

Bergers et al (1999) Science 284:808-12.

•Transgenic mouse model of pancreatic islet cell carcinogenesis

•Rat insulin promoter driving expression of SV40 T antigen oncogene which neutralises p53 & Rb

•Allows effects of treatments aimed at prevention, intervention & regression to be explored – so is better pre-clinical model

Effects of angiogenesis inhibitors on multistage carcinogenesis in the RIP-Tag model

IT = Initial tumours at start of trialPBS = saline controlBB-94 = MMP inhibitor

AGM-1470 = angiogenesis inhibitor(mechanism uncertain)

Endostatin = collagen XVIII fragment

Angiostatin = plasminogen fragment

•BB94 only effects early & mid stage tumours•So if had used correct pre-clinical model would have known this before clinical trials

Anti-angiogenic drugs show distinct efficacy profiles at different stages of tumour growth

So why did synthetic MMP inhibitors fail as anti-cancer agents?

• Trial design – MMPIs (in general) don’t work on very advanced cancers

• Inhibit not just MMPs but other metalloproteinases eg ADAMs whose existence was unknown

• MMPs can have unexpected anti-cancer roles

So why did synthetic MMP inhibitors fail as anti-cancer agents?

• Trial design – MMPIs (in general) don’t work on very advanced cancers

• Inhibit not just MMPs but other metalloproteinases eg ADAMs whose existence was unknown

• MMPs can have unexpected anti-cancer roles

High MMP8

Low MMP8

Rela

pse

-fre

e s

urv

ival (%

)

Time after primary surgery (months)

MMP 8 -ve

MMP 8 +ve

High MMP-8 predicts improved survival in breast cancer

C.Pennington,S. Porter & P. Span

Kaplan-Meier analysis of Relapse-Free Survival time of node-negative patients that were not treated with adjuvant therapy.

Over-expression of Mmp-8 suppresses B16F10 lung colonization

Backed up by mouse model

100

80

60

40

20

0

Ove

rall

su

rviv

al (

%)

100

80

60

40

20

0

Rel

apse

-fre

e su

rviv

al (

%)

Expression of ADAMTS8 and ADAMTS15 predicts survival in breast cancer

Overall survival Relapse-free survival

ADAMTS8 low

ADAMTS8 high

ADAMTS15 high

ADAMTS15 low

Porter et al 2006 Int J. Cancer 118: 1241