Bone Metastasis in Breast Cancer:Bone Metastasis in Breast Cancer:Molecular PathogenesisMolecular Pathogenesis

Maurizio LongoMaurizio LongoDip. Medicina Sperimentale, Università dellDip. Medicina Sperimentale, Università dell’’AquilaAquila

Highlights in the Management of Breast CancerHighlights in the Management of Breast CancerRoma, Domus Sessoriana, Nov. 16-17 2006Roma, Domus Sessoriana, Nov. 16-17 2006

Bone Metastasis: a Most Severe ComplicationBone Metastasis: a Most Severe Complicationof Breast Cancer of Breast Cancer

• Up to Up to 80%80% of patients with metastatic breast cancer of patients with metastatic breast cancer will develop bone metastaseswill develop bone metastases

• Long-term survivor patients with only bone Long-term survivor patients with only bone metastasis undergo metastasis undergo very poorvery poor life quality life quality

• Hyper-calcemiaHyper-calcemia

• Pain, often disabling and resistant to palliationPain, often disabling and resistant to palliation

• Pathologic fracturePathologic fracture

• (Breast carcinomas account for >50% of the met. (Breast carcinomas account for >50% of the met. cases requiring orthopedic intervention)cases requiring orthopedic intervention)

Bone-Metastatic Disease of Breast Cancer:Bone-Metastatic Disease of Breast Cancer:Important SymptomsImportant Symptoms

Bone-Metastatic Disease of Breast Cancer:Bone-Metastatic Disease of Breast Cancer:Clinical FeaturesClinical Features

• MultifocalMultifocal: at autopsy, nearly all patients demonstrate : at autopsy, nearly all patients demonstrate multiple, small “asymptomatic” bone metastasesmultiple, small “asymptomatic” bone metastases

• Preferably located to the Preferably located to the axialaxial skeleton, together with active skeleton, together with active hemopoietic marrowhemopoietic marrow

• Indolent Indolent course (e.g. remarkable progression-free periods course (e.g. remarkable progression-free periods after treatment)after treatment)

Osteolysis and Pathologic FractureOsteolysis and Pathologic Fracture

Mechanical properties are severely endangered also by “mixed” Mechanical properties are severely endangered also by “mixed” lesion (lytic/blastic)lesion (lytic/blastic)

Lytic areas

METASTASIS: a Multi-step Process (I)METASTASIS: a Multi-step Process (I)

• Detachment from tumour Detachment from tumour (dependent on E-M transition)(dependent on E-M transition)

• Transport in the bloodstreamTransport in the bloodstream(estimated <0.1% surviving this phase)(estimated <0.1% surviving this phase)

• Adhesion to endothelial cellsAdhesion to endothelial cells(mostly random*)(mostly random*)

• Invasion of host tissueInvasion of host tissue

Target-independ.Target-independ.

**DebatedDebated

METASTASIS: a Multi-step Process (II)METASTASIS: a Multi-step Process (II)

Target-dependent steps:Target-dependent steps:

1.1. ““Colonisation” of tissueColonisation” of tissue

2.2. Triggering of growthTriggering of growth(cross-talk with the host tissue)(cross-talk with the host tissue)

3.3. Independent growth of secondary tumour Independent growth of secondary tumour (only autocrine and systemic regulation)(only autocrine and systemic regulation)

The Metastasis Process: “Classical” DescriptionThe Metastasis Process: “Classical” Description

An “obsolete” model?An “obsolete” model?

Main points presently challenged of the Main points presently challenged of the “classical” description:“classical” description:

1.1. Cell detachment as a late eventCell detachment as a late event

2.2. “ “Unbridled” growth of (micro)metastasesUnbridled” growth of (micro)metastases

--> --> In fact, recent evidence obtained in In fact, recent evidence obtained in in-vivoin-vivo models shows: models shows:

1)1) single tumour cells in distant tissues during earliest single tumour cells in distant tissues during earliest development phases of metastatic tumours;development phases of metastatic tumours;

2)2) extremely slow development of many micrometastases.extremely slow development of many micrometastases.

Growth of micrometastases is aGrowth of micrometastases is atarget-target-dependentdependent process process

The Bone Micro-environment:The Bone Micro-environment:Main Cell PlayersMain Cell Players

The bone organ is also comprised of: marrow stromal cells, The bone organ is also comprised of: marrow stromal cells, hemopoietic cells, adipocytes. (Fibrous tissue may also appear.)hemopoietic cells, adipocytes. (Fibrous tissue may also appear.)

Osteoblasts and Osteoclasts: Main Features Osteoblasts and Osteoclasts: Main Features

• Osteoblasts (OBs) develop from Osteoblasts (OBs) develop from mesenchimalmesenchimal cells cells• Osteoclasts (OCs) develop from the fusion of Osteoclasts (OCs) develop from the fusion of bloodblood cells of the cells of the

immune system, the monocytesimmune system, the monocytes

• OBs are bone-residing cellsOBs are bone-residing cells• OC precursors are recruited from the bloodstreamOC precursors are recruited from the bloodstream

• OBs proliferate, OCs do not. The OC life-span is limited OBs proliferate, OCs do not. The OC life-span is limited (apoptosis can only be delayed)(apoptosis can only be delayed)

Both OBs and Ocs are polarised, very actively secreting cells:Both OBs and Ocs are polarised, very actively secreting cells:• OBs secrete bone matrix glycoproteins and collagenOBs secrete bone matrix glycoproteins and collagen• OCs secrete HCl and lytic enzymes, and OCs secrete HCl and lytic enzymes, and digestdigest collagen collagen

The Bone Remodeling Unit (I) The Bone Remodeling Unit (I)

OCs OCs OBs OBs

““Reversal” cells Reversal” cells

Resorption cone. (Early osteoblasts do not adhere to the bone Resorption cone. (Early osteoblasts do not adhere to the bone surface and proliferate actively.)surface and proliferate actively.)

The Bone Remodeling Unit (II) The Bone Remodeling Unit (II)

Resorption is fast, apposition is slow, mineralisation slower.Resorption is fast, apposition is slow, mineralisation slower.I.e. --> uncontrolled resorption can damage bone fast!I.e. --> uncontrolled resorption can damage bone fast!

The resorption activity of osteoclasts is The resorption activity of osteoclasts is potentially very harmful and is potentially very harmful and is finelyfinely regulated. regulated.

WhatWhat controls osteoclasts? controls osteoclasts?

Bone Resorption by Osteoclasts:Bone Resorption by Osteoclasts:Multiple Control LevelsMultiple Control Levels

• SystemicSystemic: hormones (incl. Vitamin D3) and metabolic hormones (incl. Vitamin D3) and metabolic signalssignals

• LocalLocal::• Secreted factors (chemokines, interleukins, Secreted factors (chemokines, interleukins,

cytokines)cytokines)• Cell-cell interactions Cell-cell interactions

Note well: at the local level, phisiological Note well: at the local level, phisiological regulation relies crucially on:regulation relies crucially on:

osteoblastsosteoblasts and their precursors. and their precursors.

ChemotaxisChemotaxisProliferationProliferationFusionFusionDifferentiationDifferentiationResorptionResorptionApoptosisApoptosis

SDF-1SDF-1M-CSFM-CSF RANKLRANKL

OPGOPG

Vitamin D3Vitamin D3PTH/PTHrPPTH/PTHrP

IL-6IL-6

Osteoclast control: Local FactorsOsteoclast control: Local Factors

Vitamin D3Vitamin D3IL-1, IL-6IL-1, IL-6

c-Fmsc-Fms

RANKRANK

M-CSFM-CSF

RANKLRANKL

OPGOPG

Osteoclast control: Cell-cell InteractionOsteoclast control: Cell-cell Interaction

Key point:Key point:

Bone met. cells are, or become*, highly Bone met. cells are, or become*, highly responsive to many signals destined to bone cells.responsive to many signals destined to bone cells.

**Or both!Or both!

What What survival survival andand growth growth signals for bone cells signals for bone cells do met. cells exploit ?do met. cells exploit ?

Micrometastasis cell originate from (actively) dividing tumour Micrometastasis cell originate from (actively) dividing tumour cells with cells with unlimitedunlimited division potential division potential

Despite this, micrometastasis show an Despite this, micrometastasis show an indolentindolent initial initial developmentdevelopment

ANALOGY:ANALOGY:

Stem cells are characterised by unlimited division potentialStem cells are characterised by unlimited division potential

BUTBUT

stem cells divide with extremely low frequencystem cells divide with extremely low frequency

1. 1. SurvivalSurvival: Stem Cell Niches: Stem Cell Niches

The “Cancer Stem Cell” Hypothesis:The “Cancer Stem Cell” Hypothesis:Tumours originate from stem/progenitor cells of the host tissueTumours originate from stem/progenitor cells of the host tissue

Normal tissuesNormal tissues Carcinomas, etc.Carcinomas, etc. Adenomas,Adenomas,sarcomas, etc.sarcomas, etc.

The Hemopietic Stem Cell Niche in Bone (I)The Hemopietic Stem Cell Niche in Bone (I)

Niche-dependent differentiation in healthy boneNiche-dependent differentiation in healthy bone

Stem Cell Niche in Bone:Stem Cell Niche in Bone:Overview of Present KnowledgeOverview of Present Knowledge

• Formed by SNO cells, an Formed by SNO cells, an osteoblasticosteoblastic sub-population sub-population

• SNO cells are:SNO cells are:• Spindle-shapedSpindle-shaped (poorly polarised / non-secreting) (poorly polarised / non-secreting)• N-Cadherin-positiveN-Cadherin-positive• CD45-negativeCD45-negative

• Formed by SNO cells, an Formed by SNO cells, an osteoblasticosteoblastic sub-population sub-population

• SNO cells are:SNO cells are:• Spindle-shapedSpindle-shaped (poorly polarised / non-secreting) (poorly polarised / non-secreting)• N-Cadherin-positiveN-Cadherin-positive• CD45-negativeCD45-negative

• SNO cells maintain self-renewing long-term stem cells in a SNO cells maintain self-renewing long-term stem cells in a semi-semi-quiescentquiescent state state

• Thus, activation of stem cells requires Thus, activation of stem cells requires detachmentdetachment from SNO from SNO cellscells

Stem Cell Niche in Bone:Stem Cell Niche in Bone:Overview of Present KnowledgeOverview of Present Knowledge

• Formed by SNO cells, an Formed by SNO cells, an osteoblasticosteoblastic sub-population sub-population

• SNO cells are:SNO cells are:• Spindle-shapedSpindle-shaped (poorly polarised / non-secreting) (poorly polarised / non-secreting)• N-Cadherin-positiveN-Cadherin-positive• CD45-negativeCD45-negative

• SNO cells maintain self-renewing long-term stem cells in a SNO cells maintain self-renewing long-term stem cells in a semi-semi-quiescentquiescent state state

• Activation of stem cells requires Activation of stem cells requires detachmentdetachment from SNO cells from SNO cells

Stem Cell Niche in Bone:Stem Cell Niche in Bone:Overview of Present KnowledgeOverview of Present Knowledge

• Detachment from the niche is favoured by various phisiological Detachment from the niche is favoured by various phisiological (e.g. chemotactic) and pathological signals(e.g. chemotactic) and pathological signals

The Cancer Stem Cell Niche in BoneThe Cancer Stem Cell Niche in BoneHypothesis:Hypothesis:

Once in the bone marrow micro-environment, Once in the bone marrow micro-environment, cancer stem cell may achieve long cancer stem cell may achieve long survivalsurvival by by

interacting with stem-cell nichesinteracting with stem-cell niches

The Cancer Stem Cell Niche in BoneThe Cancer Stem Cell Niche in BoneHypothesis:Hypothesis:

Once in the bone marrow micro-environment, Once in the bone marrow micro-environment, cancer stem cell may achieve long survival by cancer stem cell may achieve long survival by

interacting with stem-cell nichesinteracting with stem-cell niches

On the other hand, the niche will prevent cancer stem cells from On the other hand, the niche will prevent cancer stem cells from proliferate appreciably! proliferate appreciably! <-- <-- Explanation of metastasis indolenceExplanation of metastasis indolence

Additional signals can lead to further metastasis progressionAdditional signals can lead to further metastasis progression

Abnormal niche-dep. differentiation in metastasis-bearing boneAbnormal niche-dep. differentiation in metastasis-bearing bone

The Hemopietic Stem Cell Niche in Bone (II)The Hemopietic Stem Cell Niche in Bone (II)

The Initial Development of MetastasesThe Initial Development of Metastasesor:or:

««Spies in Enemy Headquarters»Spies in Enemy Headquarters»

2. 2. GrowthGrowth::

• Initial metastases (few cancer cells) Initial metastases (few cancer cells) cannotcannot rely on autocrine rely on autocrine growth-promoting (and anti-apoptosis) signalsgrowth-promoting (and anti-apoptosis) signals

• Thus, only metastasis cells responding best to available Thus, only metastasis cells responding best to available growthgrowth signals will undergo positive selectionsignals will undergo positive selection

• Of these, cells also capable of over-stimulating local bone Of these, cells also capable of over-stimulating local bone resorption will be further favoured (due to creation of larger resorption will be further favoured (due to creation of larger space for growth)space for growth)

How can an ex-epithelial cell manage to live How can an ex-epithelial cell manage to live well once free in the bone marrow?well once free in the bone marrow?

The Metastasis “Vicious Circle” (I) The Metastasis “Vicious Circle” (I)

The Metastasis “Vicious Circle” (II) The Metastasis “Vicious Circle” (II)

Cancer cells

PTHrP, etc.

Bone Matrix

BoneResorption

RankL-Rank

TGF-

IGF-1PDGF

BMPs

FGFs

ProliferationSurvival

Migration

OBsPre-OCs

OCs

IL-6, TNFα, M-CSF, PGE2

Observation:Observation:

Beside releasing same OC-genic factors as OBs, Beside releasing same OC-genic factors as OBs, various bone met. cells also express bone-cell various bone met. cells also express bone-cell

markers (e.g. BSP, OPN) and transcription factors markers (e.g. BSP, OPN) and transcription factors (e.g. Runx2).(e.g. Runx2).

Observation:Observation:

Beside releasing same OC-genic factors as OBs, Beside releasing same OC-genic factors as OBs, various bone met. cells also express bone-cell various bone met. cells also express bone-cell

markers (e.g. BSP, OPN) and transcription factors markers (e.g. BSP, OPN) and transcription factors (e.g. Runx2).(e.g. Runx2).

Just a coincidence?Just a coincidence?

• Initial metastases (Initial metastases (fewfew cancer cells) cancer cells) cannotcannot rely on autocrine rely on autocrine growth-promoting (and anti-apoptosis) signalsgrowth-promoting (and anti-apoptosis) signals

• Thus, onlyThus, only metastasis cells responding best to available growth metastasis cells responding best to available growth signals will undergo positive selectionsignals will undergo positive selection

• Of these, cells also capable of over-stimulating local bone Of these, cells also capable of over-stimulating local bone resorption will be further favoured (due to creation of larger resorption will be further favoured (due to creation of larger space for growth)space for growth)

How can an epithelial cell manage to live well in How can an epithelial cell manage to live well in the bone marrow?the bone marrow?

Result: various metastasis cells will eventually Result: various metastasis cells will eventually “resemble”* the bone-residing cells governing “resemble”* the bone-residing cells governing

bone resorption (bone resorption (i.e. OBs!i.e. OBs!))

OROR

The Met. “The Met. “OsteomimicryOsteomimicry” Hypothesis” Hypothesis

**Pre-adaptation also possiblePre-adaptation also possible

• Lin DL, Tarnowski CP, Zhang J, Dai J, Rohn E, Patel AH, Morris MD, Keller ET.Bone metastatic LNCaP-derivative C4-2B prostate cancer cell line mineralizes in Bone metastatic LNCaP-derivative C4-2B prostate cancer cell line mineralizes in vitro.vitro.Prostate. 2001 May 15;47(3):212-21. Prostate. 2001 May 15;47(3):212-21.

• Barnes GL, Javed A, Waller SM, Kamal MH, Hebert KE, Hassan MQ, Bellahcene A, Van Wijnen AJ, Young MF, Lian JB, Stein GS, Gerstenfeld LC.Osteoblast-related transcription factors Runx2 (Cbfa1/AML3) and MSX2 mediate the Osteoblast-related transcription factors Runx2 (Cbfa1/AML3) and MSX2 mediate the expression of bone sialoprotein in human metastatic breast cancer cells.expression of bone sialoprotein in human metastatic breast cancer cells. Cancer Res. 2003 Cancer Res. 2003 May 15;63(10):2631-7. May 15;63(10):2631-7.

• Zhang JH, Tang J, Wang J, Ma W, Zheng W, Yoneda T, Chen J.Over-expression of bone sialoprotein enhances bone metastasis of human breast cancer cells Over-expression of bone sialoprotein enhances bone metastasis of human breast cancer cells in a mouse model. in a mouse model. IInt J Oncol. 2003 Oct;23(4):1043-8. nt J Oncol. 2003 Oct;23(4):1043-8.

• Barnes GL, Hebert KE, Kamal M, Javed A, Einhorn TA, Lian JB, Stein GS, Gerstenfeld LC.Fidelity of Runx2 activity in breast cancer cells is required for the generation of metastases-Fidelity of Runx2 activity in breast cancer cells is required for the generation of metastases-associated osteolytic disease.associated osteolytic disease. CCancer Res. 2004 Jul 1;64(13):4506-13. ancer Res. 2004 Jul 1;64(13):4506-13.

Met. Met. OsteomimicryOsteomimicry: Some Evidence : Some Evidence

Transcriptome AnalysisTranscriptome Analysis::

Genes more expressed in bone mets versus non-Genes more expressed in bone mets versus non-bone-mets: any bone-cell markers?bone-mets: any bone-cell markers?

YES!YES!

Gene symbol Ratio bone mets vs non-bone mets

Bone metsvs primary

Ratio bone mets vs normal bone

MMP-9 32 12 2.21

ITGB3 2.0 4.4 0.63

CTSK 16 1.5 0.82

IBSP 11 31 0.25

OMD 37 3.4 0.36

RAMP-2 2.7 1.8 0.23

MEOX-2 4.7 2.8 0.37

IGF-BP5 4.3 4.4 0.91

Transcriptome AnalysisTranscriptome Analysis::The Osteomimicry SignatureThe Osteomimicry Signature

Gene symbol Ratio bone mets vs non-bone mets

Bone metsvs primary

Ratio bone mets vs normal bone

MMP-9 32 12 2.21

ITGB3 2.0 4.4 0.63

CTSK 16 1.5 0.82

IBSP 11 31 0.25

OMD 37 3.4 0.36

RAMP-2 2.7 1.8 0.23

MEOX-2 4.7 2.8 0.37

IGF-BP5 4.3 4.4 0.91

The Osteomimicry Signature:The Osteomimicry Signature:OB Genes*OB Genes*

**Shown only Shown only cSrccSrc-regulated genes-regulated genes

Gene symbol Ratio bone mets vs non-bone mets

Bone metsvs primary

Ratio bone mets vs normal bone

MMP-9 32 12 2.21

ITGB3 2.0 4.4 0.63

CTSK 16 1.5 0.82

IBSP 11 31 0.25

OMD 37 3.4 0.36

RAMP-2 2.7 1.8 0.23

MEOX-2 4.7 2.8 0.37

IGF-BP5 4.3 4.4 0.91

The Osteomimicry Signature:The Osteomimicry Signature:OCOC Genes Genes

Hypothesis (I):Hypothesis (I):

The osteomimicry “strategies”* of bone The osteomimicry “strategies”* of bone

metastasis cells relies not only on osteoblast-, but metastasis cells relies not only on osteoblast-, but

also on osteoalso on osteoclastclast-like features-like features

**Multiple osteomimetic pseudo-phenotypes possibleMultiple osteomimetic pseudo-phenotypes possible

Hypothesis (II):Hypothesis (II):

Part of the observed pseudo-osteoclastic Part of the observed pseudo-osteoclastic

phenotype of bone met. cells might accomplish phenotype of bone met. cells might accomplish

“micro-resorption”.“micro-resorption”.

Hypothesis (II):Hypothesis (II):

Part of the observed pseudo-osteoclastic Part of the observed pseudo-osteoclastic

phenotype of bone met. cells might accomplish phenotype of bone met. cells might accomplish

“micro-resorption”.“micro-resorption”.

Micro-resorption could be the trigger of the Micro-resorption could be the trigger of the

metastasis vicious circle. metastasis vicious circle.

Thank You for your attention!Thank You for your attention!