A Multigenic Program Mediating Breast Cancer Metastasis to Bone

1
A Multigenic Program Mediating Breast Cancer
Metastasis to Bone
Kang Y, Siegel PM, Shu W, Drobnjak M, Kakonen SM,
Cordon-Cardo C, Guise TA, Massague J.Cancer
Cell. 2003 Jun3(6)537-49. Leon Bernal-
Mizrachi Washington University/BJH
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Case Presentation

• 56 y/o white female diagnosed with breast cancer
  SIV (T2N1M1), metastasis to the liver 10 months
  ago.
• Biological profile ER/Pr (-), Her2 ()
• Patient started on herceptin/ Navelvine because
  of concern of alopecia. The patient achieved CR,
  reason for stopping the Navelvine and continuing
  her on Herceptin.(Fumoleau et al. 1995 Gasparini
  et al. 1994Jones et al. 1995 Romero et al.
  1994 Twelves et al. 1994Vogel et al. 1999
  Weber et al. 1995)
• Six months later she presented recurrence of the
  tumor and new metastatic disease was found in the
  hip, reason for starting the patient in
  Herceptin/Gemzar. (Oshaughenessy JA, Breast Ca
  Res Treat. 2001)
• Now tumor marker is on the rise, CT scan
  demonstrated lack of progression of the disease,
  but patient states primary is slowly growing.
• Is it a different primary ? Or has the tumor
  mutated?

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Breast Cancer and Bone Metastasis

• Frequency
• Bone is often the first, and most common, site
  for metastatic disease in breast cancer.
• Single cytokeratin-positive epithelial cells has
  been seen in bone marrow aspirates in 36 of the
  patients with stage breast cancer (SI-SIII).
• One hundred of 302 patients with N0 disease
  presented bone marrow micrometastasis (S. Braun
  et al. NEJM. 2000)
• Clonality of the Metastatic tissues
• Examination of the genetic differences between
  primary tumors, locoregional recurrences, and
  metastatic lesions indicate that, although
  clonal, many metastases show few of the genetic
  changes seen in the primary tumor, which itself
  shows significant genetic heterogeneity.
• Suggesting that the primary and metastasis may be
  more like distant cousins rather than parent and
  child (T. Kuukasjarvi, Cancer Res,1997)
• No relation between the genomic changes in the
  bone marrow cytokeratin positive cells and the
  primary tumor. (O. Schmidt-Kittler, P Natl Acad
  Sci,2003)

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Aim of the Study

• How different genes cooperate to fulfill various
  requirements for the establishment of
  tissue-specific metastases, remain important open
  questions.

5
Isolation of Metastatic Cells

• In vivo selection of highly metastatic breast
  cancer cells

105 cells
12 weeks
7 weeks
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Incidence of Bone Metastasis Generated by
Parental and various sublines of MDA-md-231
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Metastatic Effect To Bone Is A Trait Retained
During Passage In Vivo And In Vitro
Faster tumor growth in the bone but not in other
organs
The bone metastatic derived cells kept their
affinity to produce bone mets After in vitro
culture or second passage
8
Osteoclast Recruitment by highly Osteolytic Tumor
Cell Population
Cell lines with affinity to do adrenal metastasis
Cell Lines with Affinity to do bone metastasis
Higher incorporation of osteoclast into the tumor
mass
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Gene Profile Studies

• Poor prognosis gene expression did not differed
  between primary cells and highly bone metastatic
  derived cells

7/10
14/38

• It was found a bone specific signature, that was
  different from the poor prognosis profile

• Genes that were over expressed gt4 fold encoded
  cell membrane or secretory products

43/102
59/102

• Genes that were under expressed encoded
  extracellular matrix, cytoskeletan components
  and receptors, proteinases and MHCII

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Functional Studies Of Candidate Genes Supposed to
Promote Metastasis

• Stable transfectants of IL-11 did not show an
  enhanced bone metastasis formation

• Stable The combination of IL-11 and OPN
  significantly augmented the incidence of bone
  metastasis
• Himmunohistochemical studies of the resulting
  lesions revealed the presence o f multiple TRAP
  () cells

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Functional Studies of Candidate Genes Supposed to
Promote Metastasis

• Stable transfectants of CXCR-4 cause a limited
  but significant increase in metastasis formation
  whereas CTGF did not
• Triple transfectants overexpressing either
  IL-11/OPN either CXCR-4 or CTGF presented
  increase of rate and incidence of bone mets.

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TGFb Activates Bone Metastasis Genes IL-11 And
CTGF

• Recent observations showed that in breast cancer
  cells TGFb and smad turn from a tumor suppressor
  into a tumor progression factor
• ( Chen et al., 2001 and Derynck et al.,2001).
• Parental MDA-MB-231 and derived cells when
  culture with TGFb demonstrated an elevated
  secretion of PTHrP ( but not increase levels of
  mRNA were seen).
• The levels of PTHrP were not different between
  parental cells and derived cells

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TGFb Activates Bone Metastasis Genes IL-11 And
CTGF

• Parental MDA-MB-231 were
• incubated with TGFb-1
• demonstrated enhanced
• expression of the already
• elevated levels of IL-11 and
• CTGF.
• TGFb/smad pathway induction of IL-11 and CTGF may
  participate in a bone metastasis cycle driven by
  a paracrine TGFb

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Evidence In Vivo of Preexisting Metastatic Cells

• Analysis of signature in culture cells and
  selection

• Cells inoculated in athymic mice demonstrated a
  more aggressive development of bone metastasis
  signature.

The number of genes expressed correlated with
the time of bone met development
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Evidence In Vivo of Preexisting Metastatic Cells
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Evidence In Vivo of Preexisting Metastatic Cells

• The proportion of cells expressing each of the
  metastasis genes tested was dramatically
  increased in the 1833 and 2287 cell lines
  compared to the parental population

• Comparative genomic hybridization (CGH) analysis
  on the parental
• MDA-MB-231 demonstrated that the chromosomal
  profile of these
• populations is highly aberrant compared to
  normal human cells, but the
• profile of the subpopulations was very similar
  despite the marked
• differences in metastasis gene patterns and
  metastatic activities.

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Conclusion and Bone Metastatic Model
Conclude that the tumor-derived MDA-MB-231
population contains natural variants with a
bone-metastasis gene signature and high
metastatic activity to bone these variants
become selected in vivo by virtue of their high
propensity to form fast-growing bone
metastases. The authors propose that the
poor-prognosis gene expression signature enables
the emergence of metastatic cells, whereas the
bone metastasis gene expression signature
identified here executes the metastatic potential
of such cells.
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Questions to be Answer

• No studies were done in primary tumors after
  transplant of the cells.
• Important point because it will allow us to
  determine if the primary tumor cell line is a
  pluripotential or if the primary tumors are
  complex system of different cell lines with a
  predetermine destiny and function.
• There was no analysis about hormone receptor
  status and Her-2 status.
• Important because it can elucidate selective
  responses to distinct therapeutic approaches