Metastatic Breast Cancer: Seed or Soil

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Metastatic Breast Cancer Seed or Soil?

• Katherine Weilbaecher, M.D.
• Assistant Professor in Medicine and Cell Biology
  and Physiology
• Division of Oncology

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Metastasis

• Dissemination of cancer away from the primary
  site and formation of new tumors
• As opposed to local invasion
• Lymph Node- via lymphatics
• Visceral-the most deadly (liver,lung,adrenal)
• Bone/bone marrow- specialized
• Brain- issues of blood/brain barrier
• Median survival time for patients with metastatic
  breast cancer is 2-4 years (long term survival is
  possible).

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Metastasis

• The cause of death in 90 of cancer patients
• The cause of significant pain and suffering
• Signifies that a cancer is usually incurable
• Need for effective systemic therapy- local
  therapies usually not effective
• Least well understood of all stages of
  carcinogenesis

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At Presentation
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Only 50-70 of patients with breast cancer will
be cured
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Bone and Liver Metastases
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Occult Tumor Cells in Bone Marrow Correlate with
Poor Prognostic Factors
Overall Survival
Gerber et al, JCO 19(4)960-971, 2001
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Treatment of Breast Cancer- Main Goal is to
Prevent Metastasis

• Local therapies Surgery and Radiation
• Systemic Therapy Adjuvant chemotherapy and/or
  hormone therapy increases cure rate by 2-10 (but
  everyone gets treated)
• Other therapies Bisphosphonates, angiogenesis
  inhibitors, Cox-2 inhibitors, Her-2/EGFR1
  inhibitors, immune stimulators, Diet-low fat,
  high fiber, red wine, vitamins, laughter therapy

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Stages of Tumor Metastasis
Mundy. Nat. Rev. Cancer. 20022584-593.
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Genes Involved in Metastasis

• Integrins- receptors involved in cell-
  extracellular matrix (ECM) interactions
• Matrix Metalloproteases (MMP) - involved in ECM/
  adhesion molecule degradation
• Cadherins- receptors involved in cell-cell
  interactions
• Growth factors/receptors

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Metastasis Formation is Highly Inefficient

• 0.01 of highly metastatic cells form tumor foci
  after intravenous injection.
• 1cm3 size tumor has 109 cells. 106 cells will be
  shed into the circulation each day.
• 95 of all breast cancer patients have
  detectable circulating tumor cells.
• Metastases from renal cell carcinoma, melanoma,
  colon cancer and breast cancer can lie dormant
  for 15-20 years
• Angiogenesis inhibition, immune status, hormonal
  changes, new mutations.

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Seed and Soil Hypothesis

• In cancer of the breast, the bones suffer in a
  special way, which cannot be explained by any
  theory of embolism alone.
• when a plant goes to seed, its seeds are carried
  in all directions, but they can only grow if they
  fall on congenial soil.
• Stephen Paget

Paget. Lancet. 1889 1571-2.
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Incidence of Metastases to Bone
Bone Lesions ()

• Myeloma 95-100
• Breast 65-75
• Prostate 65-90
• Thyroid 60
• Bladder 40
• Lung 30-40
• Renal 20-25
• Melanoma 14-45

350,000 patients in US die with bone metastases
each year
Mundy. Nat. Rev. Cancer. 20022584-593.
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Complications of Skeletal Metastases

• Pain
• Analgesics, constipation, confusion, radiation
• Pathologic bone fracture
• Pain, immobility, surgery, radiation
• Spinal cord compression
• Hypercalcemia
• Anorexia, confusion, constipation

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Skeletal Metastases

• Cancer cells require normal host cells to
  metastasize to bone
• Osteoclast activation is central bone metastases
• Therapies targeted to block osteoclast
  development and function can block bone metastases

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Osteo Lytic
Osteo Blastic
Mundy. Nat. Rev. Cancer. 20022584-593.
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Bone Formation
Estrogen
Osteoblast
T
Glucocorticoids
M-CSF RANKL
T
Estrogen
Glucocorticoids
Osteoclast
T
Bisphosphonates
Bone Resorption
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Tumor Mediated Bone Invasion
TGFb, IGFs,BMPs FGFs, PDGF
PDGF VEGF FGFs IGFs TGFb
RANKL IL-6 BMPs SDF-1 MCP-1 MIP-1a
Endothelin 1
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Osteoclast Inhibitor-OPG Prevents Tumor
Associated Bone Destruction
Honore et al. Nat Medicine 2000, 6521-528.
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OPG does not not effect tumor growth in bone
Honore et al. Nat Medicine 2000, 6521-528.
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OPG decreases Astrocyte Hypertrophy in spinal
cord
Honore et al. Nat Medicine 2000, 6521-528.
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OPGL/RANKL and Tumor Mediated Osteolysis and Pain
HOW?
Thompson and Tonge, Nature Medicine.
20006504-506.
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Bisphosphonates- Inhibitors of
Osteoclastogenesis and Function

• Etidronate 1.0
• Clodronate 10
• Pamidronate 100
• Alendronate 1000
• Ibandronate 10,000
• Zoledronic acid 100,000

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Pharmacokinetics of Bisphosphonates

• Rapid absorption to bone
• 60 of the dose retained in the skeleton
• 50 of the dose still in the skeleton after 6
  months
• Gastrointestinal absorption is poor
• did not exceed 5 of the dose
• amount available to bone was 0.1-0.2 after an
  oral dose
• Renal excretion (unmetabolized)
• what is not bound to bone is excreted within
  approximately 24 hours

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Pamidronate Decreases Skeletal Complications and
Pain in Women with Breast Cancer Osteolytic
Metastases
Skeletal Complications
Pain
N185 Pamidronate N195 Placebo
Hortobagyi et al. NEJM. 19963351785-1792.
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Tumor Mediated Bone Invasion
TGFb, IGFs,BMPs FGFs, PDGF
PDGF VEGF FGFs IGFs TGFb
RANKL IL-6 BMPs SDF-1 MCP-1 MIP-1a
Endothelin 1
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Left Cardiac Ventricular (LV) injection of Tumor
Cells

• Current models of bone metastases involve
  intracardiac (arterial) injection of tumor cell
  lines.

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Chambers et al. Nat Rev Cancer, 2002 2563.
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Dominant Negative TGFbRII Expression in
MDA-MB-231 Breast Cancer Cells Impairs Tumor
Induced Osteolysis
PTHrp
Yin et al. JCI, 103197-206 (1999)
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PTHrp Overexpression Restores Tumor Osteolysis in
DN-TGFbRII MDA-231 Mutants
Yin et al. JCI, 103197-206 (1999)
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Tumor Production of PTHrp Induces Osteolytic
Destruction
Breast Cancer Cell
Käkönen and Mundy Cancer.2003 97,S3834-839.
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Human ER- breast cancer cell line MDA-MB-231
develops higher metastatic capacity after in
vivo passaging
Kang et al. Cancer Cell. 2003 3 537-549.
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Fidler. Nature New Biol, 1973242148-149.
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Parental MDA cells already Express Poor-prognosis
gene set
Parental MDA and Strong Metastasis clones have
different Gene expression patterns
Kang et al. Cancer Cell. 2003 3 537-549.
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Human Breast Cancer Cell Line MDA-MB-231
Transfected with Metastasis Genes Increase
Bone Tropism
Kang et al. Cancer Cell. 2003 3 537-549.
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Individual MDA cells from parental line have
highly metastatic Phenotype and genotype
Kang et al. Cancer Cell. 2003 3 537-549.
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Dynamic heterogeneity
Equilibrium between Highly metastatic
(varient) And non-metastatic (majority)
5x105
1x107
Hill et al. Science. 1984 224998-1001.
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Bone Tropism Mediated by Metastasis Genes
Kang et al. Cancer Cell. 2003 3 537-549.
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Gene expression profile nearly identical between
primary cancer and lymph node metastasis

• mRNA from human breast tumors from primary tumor
  and lymph node metastasis in same patients were
  collected
• Gene expression profiles were almost identical
  suggesting that the molecular program of a
  primary tumor is generally retained in its
  metastases.

Perou et al. Nature. 2000, 406747-752.
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Bone Marrow Micrometastasis have fewer
chromosomal abnormalities than primary tumor by
CGH analysis
Schmidt-Kittler et al. PNAS. 2003100(13)7737-42
.
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Bone Marrow Micrometastasis from M0 have fewer
Chromosomal abnormalities than M1 patients by
CGH analysis
M0-black M1-white
Schmidt-Kittler et al. PNAS. 2003100(13)7737-42
.
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Animal Models of Metastasis- Call for Research!

• Few spontaneous models of metastases- her2-
  neu,TRAMP, RipTag2
• Tumor cell line injection models- IV, LV
• Orthotopic transplantation of tumors
• Problems of species barrier and immunocompetance

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Proposed Role of Host Cell b3 Integrin Expression
in Metastasis
Endothelium
Platelet
b 3
b 3
b 3
b 3
Osteoclast
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Genes Critical to Osteoclast Development
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Hypothesis

• Beta 3 integrin expression on host cells is
  critical to tumor bone invasion.
• Tumor bone invasion will be impaired in the b3-/-
  mouse.

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INTEGRIN FAMILY
R. Hynes Cell .2002110 673-687.
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Matrix binding promotes integrin clustering and
association with the cytoskeleton
Motility Survival Proliferation Shape change
Hood and Cheresh, Nature Reviews Cancer.2002
291-100.
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src
avb3 Integrin
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b3 Integrin-/- have Dysfunctional Osteoclasts
and Platelets

• b3-/- develop Osteosclerosis with age
• b3-/- OCs have defective actin rings, defective
  ruffled border and defective bone resorption
• b3-/- have defective Platelet Aggregation
• increased bleeding time, cutaneous and
  gastrointestinal bleeding

Hodivala-Dilke et al. JCI, 1999,
103229-238. McHugh et al. JCI, 2000,
105443-440.
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Left Cardiac Ventricular (LV) injection of Tumor
Cells- Bone Invasion assay

• Current models of bone metastases involve
  intracardiac injection of tumor cell lines.
• In this study- we used the B16-F10 murine
  melanoma cell line

Yoneda et al. Cancer 2000, 882979-88. Arguello
et al. Cancer Res. 1988,486876-6881.
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b3-/- Mice Are Protected from Bone Invasion
b3/
b3-/-
a
b
c
Bakewell et al. PNAS. 2003 10014199-204.
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Did the Tumor cells get to the bone marrow?
Experimental Approach Directly inoculate B16
tumor cells into the bone marrow cavity.
Intra-tibial Injection
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Decreased Bone Loss in b3-/- Mice After B16 IT
Injection
b3-/-
b3/
a
b
P
TTumor SSaline
T
S
S
T
b3/
b3-/-
Bakewell et al. PNAS. 2003 10014199-204.
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Intra tibial- conclusions

• b3-/- bone environment can support growth of B16
  cells
• B16 does not induce trabecular bone destruction
• B16 cells do not directly resorb bone

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Are there anatomic/microenvironment defect(s) in
b3-/- mice that impair tumor entry into bone?

• Experimental Approach
• Bone Marrow Transplantation to restore
  hematopoeitic cell function (osteoclast/platelets)
  
• Possible rescue of endothelial cell function

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Bone Marrow Transplantation Assay

• Mice are lethally irradiated with 950 rads
• 5 x 106 bone marrow injected by tail vein
• 2-3 weeks post-BMT mice are injected via
  left-ventricle with 105 B16 cells
• 14 days post-LV. injection mice are sacrificed
  and metastases examined

WTWT
FWT
WTKO
Bakewell et al. PNAS. 2003 10014199-204.
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Bone Marrow Transplantation Normalizes b3-/-
Bleeding Time and b3-/- Osteoclast Spreading
3 weeks after Transplant
b3-/-
WT
WT b3-/-
BMMs cultured for 6 days with GST-RANKL and
mM-CSF
5 mice/group
Bakewell et al. PNAS, 2003, 10014199-204.
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Bone Marrow Transplantation Restores Bone
Metastases in b3-/-
Percent Metastases after Bone Marrow Transplant
n 10
n 13
n 7
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Do the hematopoetic lineage osteoclasts play a
critical role in faciliting B16 entry and growth
into bone?

• Experimental Approach
• Perform bone metastasis assay in an osteoclast
  defective src-/- mouse with normal platelet
  function
• Src-/- osteoclasts are TRAP multinucleated but
  do not form a ruffled border, have abnormal
  motility and resorption

Soriano et al. Cell. 1991 64693-702
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Src-/- have Dysfunctional Osteoclasts and
Grossly Normal Platelet Function
Bakewell et al. PNAS. 2003 10014199-204.
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Src-/- Mice are Not Protected from Metastases
src/
src-/-
n8
n6
n8
n6
Bone
Visceral
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Src-/- mice are protected from tumor associated
bone loss
Bakewell et al. PNAS, 2003, 10014199-204.
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Src-/- conclusions

• B16 do not directly destroy trabecular bone
• Src expression in osteoclasts facilitates tumor
  associated bone destruction
• Osteopetrosis and defective osteoclast function
  do not prevent tumor entry into bone marrow and
  did not impair tumor growth in bone marrow

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Proposed Role of Host Cell b3 Integrin Expression
in Metastasis
b 3
b 3
b 3
b 3
Mundy. Nat. Rev. Cancer, 2002, 2584-593.
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Will inhibition of platelet beta 3 integrin
function prevent tumor entry into bone?

• Experimental Approach
• Inhibitor of murine platelet GP2b3a (aIIbb3)
• Half life is 3 hours- but platelet aggregation
  abnormal for at least 8 hours
• requires oral gavage
• 100mg/kg induces complete blockade of platelet
  aggregation at 30 minutes

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Thrombosis and Cancer

• Association noted for 140 years (Trousseau)
• Incidence of clinically important thrombosis is
  5-60 for solid tumor malignancies
• Twice as likely to have post-surgical DVT
• Hematologic malignancies have significantly lower
  risk of thrombosis
• Occult cancer found in 6-16 of patients with DVT
• Improved survival in cancer patients treated with
  Heparin in small randomized trials
• Tumors express
• Tissue factor like activity and thrombin

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GPIIb-IIIa (aIIbb3) mediates platelet aggregation
ADP
Fibrinogen, vWf
Thrombin
GP IIb-IIIa
GP Ib-V-IX
Fibrinogen
vWf
GPIIb-IIIa antagonist
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2.5 days of aIIbb3 Inhibitor Therapy Decreases
Bone and Visceral Metastases
p0.0013 p0.0125
-5 doses Inhibitor q12hr starting 30 min prior to
B16 LV Injection -14 day analysis of Metastasis
(3 separate experiments)
Bakewell et al. PNAS, 2003, 10014199-204.
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b3-/- Platelets Do Not Form Large Tumor/Platelet
Clumps
WtINH (single cell enlarged image)
WT Platelets B16
b3-/- Platelets B16
WT PLTINH B16
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Conclusions

• Host cell beta 3 integrin expression facilitates
  metastases in this animal model
• B16 Tumor induced bone destruction requires
  functional osteoclasts.
• Src and beta 3 mediated Osteoclast resorption
  required and possible drug targets
• B16 tumor growth in bone marrow does not require
  functioning host src or beta 3 expression
• Why are metastases to bone in bisphosphonate
  treated animal decreased? (non-OC effect)
• Platelet beta 3 integrin expression facilitates
  B16 tumor metastasis to bone (and viscera?)
• How do platelets interact with metastatic tumor
  cells or end organ sites?

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Conclusions

• Metastasis requires dissemination and growth in
  new environment
• Theories of Metastasis still in debate
• Orderly progression of accumulated genetic
  mutations (Vogelstein model)
• Dissemination and then new acquisition of
  mutations
• Cancer stem cells?
• Tumor-Host cell interactions are critical

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Future Directions in Metastasis Research

• Study both Seed and Soil
• Need better models
• Need to understand specific pathways
• Targeted therapies- specific to site of
  metastasis and stage of metastases (micro versus
  macro)
• Role of Host tissues platelets, immune system,
  nutrition
• Clinical trials in high risk patients