Dual%20Role%20of%20CD99%20as%20oncosuppressor%20or%20oncogene%20in%20osteosarcoma%20and%20Ewing

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Dual Role of CD99 as oncosuppressor or oncogene
in osteosarcoma and Ewings sarcoma

• Katia Scotlandi
• Istituti Ortopedici Rizzoli

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MIC2 gene

• Pseudoautosomic region of chr. X and Y.
• It does not belong to any known family of
  proteins
• It has been recognized only in primates,
  indicating a high level of evolutional diversity

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CD99

• Membrane protein of 32KDa with a high level of
  glycosilation
• Extracellular domain of 100 aa transmembrane
  domain and a short intracellular C-terminal
  domain of 38aa
• it has a hairpin shape anchored by two flexible
  loops. The cytoplasmic domain of CD99 proteins
  might only have a rigid structure when bound to
  interacting partners

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CD99

• Physiologic expression
• Ubiquitously expressed at low levels
• High expression in the hematopoietic system
  (particularly CD4CD8 T cells), pancreatic isle
  cells, ovarian cells and Sertoli cells of the
  testis

HSC Differentiated cells
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CD99
Physiologic functions
Cell-Cell and ECM Cell Adhesion ? Morphology
Apoptosis ? Differentiation positive selection of
corticotimocytes
Proliferation T-helper
Transendothelial migration of lymphocytes
during inflammation
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CD99

• Expression in tumors
• High expression in some pediatric tumors
• ? Ewings sarcoma and acute lymphoblastic
  leukemia
• Low or null expression in malignancies but high
  expression in benign lesions (stomach cancer and
  endocrine pancreatic neoplasia).

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CD99 is expressed in Osteoblasts
Primary Osteoblasts in cell culture
Lining cells Mature Osteoblasts
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and reduced in osteosarcoma
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CD99 is repressed by RUNX2 Bertaux, K. 2005
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RUNX2 in OSA

• 6p21 is amplified in most OSA

RUNX2 expression correlates with expression of
the early markers of OBL differentiation such as
ALP, COL1A1, OP r 0.7, plt0,0001
and shows inverse correlation with CD99
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CD99 acts as an oncosuppressor

• Forced expression
• induces
• Reduction of growth in soft agar
• Reduction of cell migration
• Increase cell-cell and ECM-cell adhesion
• Manara MBC 2006

U2/CD99
U2 OS
U2 OS
U2/CD99
U2/CD99
U2 OS
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• Forced expression of CD99 in OSA cells inhibits
  their metastatic potential

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CD99 acts as an oncosuppressor

• Increase OBL differentiation

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CD99 modulates gene signature of OSA cells by
inducing upregulation of pathways involved in OBL
differentiation
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• In osteosarcoma CD99 appears as a positive
  mediator of osteoblast differentiation and it
  reverts the malignant potential of the cells.

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Silencing of CD99

• Transient silencing of CD99 reduces growth in
  soft agar and cell migration (Kreppel M, 2005)

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Stable silencing of CD99
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Stable silencing of CD99

• Inhibits
• Cell growth
• Migration
• Metastatization

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Stable silencing of CD99

• Inhibits
• Cell growth
• Migration
• Metastatization

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Stable silencing of CD99

• Inhibits
• Cell growth
• Migration
• Metastatization

Incidence No. of
Time of bone mets
appearance TC-71
5/10 (50) 12 49 ?
3 TC/pCD99siRNA 23 0/10 -- --- TC/pCD99siRN
A 24 0/10 -- --- TC/CTRsiRNA 4/10 (40)
8 72 ? 6

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Stable silencing of CD99 induces neural
differentiation
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Stable silencing of CD99 induces neural
differentiation
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• In Ewings sarcoma, CD99 appears as a negative
  regulator of neural differentiation and favours
  the malignant potential of the cells

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CD99 acts coupling with caveolin-1

• CD99 and Caveolin-1 co-localize

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CD99 acts coupling with caveolin-1
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Caveolin-1

• major protein component of caveolae. Forms an
  hairpin-like structures with both the N and C
  terminal oriented towards the cytoplasm
• caveolae are more abundant in terminally
  differentiated mesenchymal cells, including
  adipocytes, endothelial cells, fibroblasts and
  osteoblasts
• Cav-1 has been described as either a tumor
  suppressor or a tumor promoter, depending on
  tumor type and/or stage

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Caveolin-1 acts as oncosuppressor in osteosarcoma

• Expression of Cav-1 modulate
• anchorage-independent growth
• Migration
• Invasion
• Metastatic ability

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Caveolin-1 acts as oncosuppressor in osteosarcoma

• Expression of Cav-1 modulate
• anchorage-independent growth
• Migration
• Invasion
• Metastatic ability

0 h
18 h
U-2/anti Cav58
U-2/Cav75
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Caveolin-1

• Expression of Cav-1 modulate
• anchorage-independent growth
• Anoikis
• Migration
• Invasion
• Metastatic ability

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Caveolin-1

• Expression of Cav-1 modulate
• anchorage-independent growth
• Anoikis
• Migration
• Invasion
• Metastatic ability

gt 250
0
2
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Met-transformed osteoblasts and osteosarcoma
samplesshow lower Cav-1 expressionthan normal
osteoblasts.
Caveolin-1
median values 1,48 (range 0.06-14.6 n37) in
OS 4,57 (range 4.2-6.1 n3) in OBL P0.03
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Caveolin-1

• No effects on in vitro cytotoxicity of the
  cells
• Its overexpression
• Decreased MAPK, Akt and c-src phosphorylation

Event-free and overall survival of 37
osteosarcoma patients according to Cav-1
expression
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Caveolin-1 is highly expressed in Ewings sarcoma

• Cav-1 is a target of EWS/Fli1 (Tirado OM Cancer
  Res 2006 Hu-Leiskovan S Cancer Res 2005)
• is necessary for tumorigenesis (Tirado OM Cancer
  Res 2006) loss of Cav-1 inhibited soft-agar
  growth and tumorigenicity

However, CD99siRNA cells have high levels of
cav-1
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Cav-1 phosphorylation could affect cav-1 functions
P-cav1 provides a docking site for ptyr binding
protein, particularly SH2-containign molecules,
and serves as a positive regulator of cell
signalling. It increases soft-agar growth and
motility ? Prostate cancer cells
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• In Ewings sarcoma CD99 silencing represses
  p-cav1

TC-71 siCTR siiCD99-23 siCD99-24 siCD99-25
BRZ siC9936
Our working hypothesis is that by repressing
p-cav-1 that in some models compete with the
caveolin scaffolding domain and binding with
signalling proteins, CD99 silencing reintroduces
the oncosuppressor role of cav-1. Indeed when
CD99 is abrogated cells showed lower level of
Akt, MAPK and c-src phosphorylation
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CD99

• Far from being a simple diagnostic marker, CD99
  regulates crucial biological processes in
  osteosarcoma and in Ewings sarcoma.
• Elucidation of its mechanisms of action will
  unravel new prognostic and therapeutic targets.

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F. Nardi
D. Zambelli
A. Rocchi
M. Zuntini
L. Cantiani
MC. Manara
M. Sciandra
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• Collaborators
• P.Picci, M. Serra, G. Nicoletti, F. Bertoni and
  P. Bacchini, IOR, Bologna
• P-L Lollini, P. Nanni, Università di Bologna
• MF. Di Renzo, IRCS Candiolo, Torino
• M.P. Colombo, INT, Milano
• G. Arancia, ISS, Roma
• A. Bernard, INSERM 574, Nice France
• Mordechai Liscovitch, Dept. Biological
  Regulation, Weizmann Institute of Science, Israel
  

AIRC EU projects PROTHETS and Eurobonet
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