Centro di
Oncobiologia Sperimentale
University of Palermo
Ida Pucci-Minafra1,2 , Nadia Ninfa Albanese1, Patrizia Cancemi1, Gianluca DiCara1, Maria Rita Marabeti1,
Francesca Costantini1, Salvatore Minafra1,2
1Dipartimento
di Oncologia Sperimentale e Applicazioni Cliniche (DOSAC), Università di Palermo, Italy.
2Centro di Oncobiologia Sperimentale (COBS), Casa di Cura di Alta Specialità La Maddalena, Palermo, Italy.
8701-BC cells colture with
Hypoxic condition
RPMI+FCS 10%
RPMI+FCS 10%
Breast cancer cells 8701-BC
Breast cancer cells 8701-BC
72h
O2=20%
8701-BC breast cancer cells
O2=2%
4gg
3gg
72h
Hypoxic
8701-BC lysate
Normoxic
8701-BC lysate
Plastic
8701-BC lysate
Type I Collagen
Type IV Collagen
2.5
TYPE I
DEC C2
Type OF/LB
8701-BC lysate
TYPE IV
TYPE V
M ean Normoxia
OF coll
M ean Hypoxia
60
O.D.
1.5
40
1.0
20
0.5
Cells grown on different collagen substrates
Cells exposed to hypoxia
0.0
0
48 h
4 gg
16h
7 gg
1.2
1.2
Control
Modulated proteins
40h
72h
Control
5 g/ml
7.5 g/ml
DEC-C3
DEC- C2
10 g/ml
-35%
O.D.
O.D.
DEC C3
Expressing DCN core
lacking the GAG side
chains.
Type V Collagen
Type OF/LB Collagen
2.0
24 h
Biological effects
of different collagen substrates
on Breast cancer cells
Type V
8701-BC lysate
80
PLASTIC
(1) Pucci-Minafra I, Fontana S,Cancemi P,Basiricò L,Caricato S,Minafra S. Proteomics 2002, 2, 919-927.
(2) Pucci-Minafra I, Cancemi P, Fontana S, Minafra L, Feo S, Becchi M, Freyria AM, Minafra S. Proteomics 2006, 6,
2609-2625.
(3) Pucci-Minafra I, Cancemi P, Di Cara G, Minafra L, Feo S, Forlino A, Tira ME, Tenni R, Martini D, Ruggeri A,
Minafra S. Connect Tissue Res. 2008, 49, 30-41.
66%
4gg
3gg
4gg
3gg
Type IV
8701-BC lysate
Type I
8701-BC lysate
8701-BC
Cells Plastic
Plastic
8701-BC Cells
4gg
3gg
4gg
3gg
Minafra et. Br. J.Cancer,
60, 185-192, 1989
0.6
Proliferation assays of
normal and treated cells
The turn-over of extracellular matrix is a physiological process, that in normal
conditions and in wound healing responds to spatial and temporal regulatory
mechanisms, involving several cell-matrix interaction pathways. Profound changes
occur, both at cellular and extracellular levels, during the progression of various forms
of invasive carcinomas. These include a progressive loss of originally stationary and
polarized epithelial phenotype, and the gain of an apolarized and motile phenotype by
neoplastic cells. Concurrently, the collagen architecture of the host stroma surrounding
the primary tumor undergoes extensive fragmentation and derangement, often adjacent
to region of extensive fibril deposition. In this context, ECM components, neoplastic
and host cells and a variety of active factors play a dynamic role in the progression of
the cancer.
In addition, increased tumor size and consequently compromised microcirculation,
induce neoplastic cell adaptation to low oxygen tension and to nutrient-deprivation.
These conditions lead to activation of several pathways that have permissive or
restrictive effects on the angiogenic processes and on the propensity of neoplastic cells
to form metastasis. However the effects of individual components of the
microenvironment on cancer cell growth and spreading remain largely unknown and
deserve further investigations. To this aim, we have used in “vitro” models of human
breast cancer cells (8701-BC) exposed to different collagen substrates and to a low O2
tension, to reproduce, at least in part, primary tumor microenvironment. Neoplastic
cell responses have been evaluated through the proteomic approach (1-3). The
obtained proteomic profiles were compared with that of cells alternatively exposed to
the influences of different ECM molecules (type I, type IV, type V, the oncofoetal
collagen, ad decorin) or under conditions of hypoxic stress (oxygen tension 2%).
Interestingly, we observed changes in cell morphology and in cell proliferation rate, as
well as in certain protein clusters, suggesting that the microenvironment may emanate
influences of opposing signal, which may contribute in directing the neoplastic cells
towards a more or less aggressive phenotype.
0.6
34%
Unmodulated proteins
I
39%
ACTB fr
AK1C3
ANXA2
ANXA4 a
ATPB
HSP27 a
LEG1 b
NDKB
NTF2
PGK1 a
S10A6
S10AB b
TPM4 a
VIME c
IV
6%
Clusterization of proteins
variably modulated
by different substrates
V
22%
OF
33%
37%
13%
TBB5 a
TYB4
IV
OF
I-IV-V
50%
I-IV-OF
30%
IV-V-OF
5%
I-IV
71%
Cells exposed to exogenous decorin
0
24h
48h
4d
5d
6d
7d
24h
48h
4d
5d
6d
7d
Proteomic and genomic modulation
induced by decorin transfection on
neoplastic cells
I
22%
I-V
18%
Decorin-transfected cells
0
V
28%
I-OF
11%
ANXA1 sf1
ANXA1 sf2
ANXA4 b
ECHM
EFTU
HSP60 a
HSP60 b
PPIA d
ACON a
ACTG
AK1BA a
ALDOA b
ALDR b
ANXA1 a
B2MG
ENOA a
G3P2 a
K1C9
MDHM a
TCPZ
Scanning Electron
Microscopy
Substrate-specific protein classes modulated are:
•Metabolic enzymes;
•Molecular Chaperones/heat shock proteins;
•Cytoskeleton and associated proteins;
•Calcium binding proteins.
I-V-OF
15%
Total trancripts analyzed
264
Total transcripts modulated
33
81 PROTEINS ARE APPARENTLY AFFECTED BY DECORIN TRANSFECTION
12,50 %
Up-regulated Genes
8
3,00 %
Down-regulated Genes
25
9,50 %
2
18 PROTEIN SPOTS SHOW INCREASED INTENSITY IN C2 AND C3 CLONES
15 PROTEIN SPOTS SHOW DECREASED INTENSITY IN C2 AND C3 CLONES
8701-BC WT
1,8
DEC-C3
700
DEC-C2
1,6
600
1,4
Espression levels of Breast cancer key-genes
Normoxia
6
5
4
3
2
1
0
200
1,2
1
0,8
22 PROTEIN SPOTS
UNIQUELY EXPRESSED
BY 8701-BC CELLS
26 PROTEIN SPOTS
UNIQUELY
EXPRESSED IN C2
AND C3 CLONES
0,6
100
0,4
TRIP13
TMEFF1
SMC4L1
SEC14L2
SERPINE1
PTDSS1
RAB27B
PRKCG
PSMD7
PRC1
PRKCB1
PPTRM1
PECI
PFKP
NUSAP1
MLF1IP
MYBL2
KRT18
MCCC1
GCN1L1
GBE1
FBX05
ARMC1
CTNNB1
CIRBP
CENPF
CCND1
BAX
BMO39
ATM
0,2
0
DEC-C2
ENO
40h
ENO
16h
AKAP1
0
GAPDH
40h
CTK 8 40h
MMP9
40h
ErbB2
40h
ErbB2
16h
c-myc
40h
c-myc
16h
0
MMP9
16h
1
MMP2
40h
2
MMP2
16h
3
Hypoxia
Effects of Hypoxia on
Breast cancer cells
ASPM
5
4
3
2
1
0
3,5
3
2,5
2
1,5
1
0,5
0
4
Normoxia
300
ASNS
Hypoxia
Normoxia
GAPDH
16h
Hypoxia
CTK 8 16h
Normoxia
400
Metabolism
trascripts
Cytoskeletal
trascripts
V im 16h
Hypoxia
Metalloproteinase
trascripts
V im 40h
Tumoral marker
trascripts
% VOLUME OF SPOTS
500
Proteome Modulation on hypoxic condition
COF1 a
TOT identified
137
UP >30%
34 (24.8%)
DOWN>30%
15 (10,9%)
Total modulation
49 (35.7%)
Mean Hypoxia
EF1b
Mean Normoxia
TPM4 a
GRP78 a
ALDR b
AK1BA a
PRDX6
LDHA
THIOm