andrea anichini human tumors immunobiology unit fondazione irccs istituto … · 2016. 3. 22. ·...
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Control of melanoma dedifferentiation program and immune escape by NFATc2
Andrea Anichini Human Tumors Immunobiology Unit
Fondazione IRCCS Istituto Nazionale dei Tumori, Milan
-
Background 1. Melanoma dedifferentiation
Consequences
+ - Intrinsic and acquired resistance to
MAPK inhibition
+ -
Promotion of immune escape and of tumor infiltration by myeloid cells
+ -
Upregulation of stemness (CD271) and dedifferentiation (AXL) markers
+ - Shift from a proliferative
to invasive gene programs
MITF + -
MITF-regulated genes, pigmentation and MDAs + -
Definition
Intrinsic regulators of melanoma dedifferentiation
ZEB1, TWIST1 In cooperation with oncogenic BRAF
Caramel, Cancer Cell 2013
Extrinsic regulators of melanoma dedifferentiation
IFN-g, NGF Furuta, J. Invest Dermatol. 2014
Drug exposure, hypoxia, and nutrient starvation
Ravindran Menon, Oncogene 2014
TNF-a
-
CD3
MITF b catenin
Loss of MITF in-vivo in the absence of a T cell infiltrate
-
Two types of melanomas with distinct MITF profiles
SOX10 / MITF AP-1 / TEADS
Master regulators of distinct transcriptional states
Proliferative gene network
Invasive gene network
MITF+ melanomas Tissue section Cell line
Pt#14
Pt#8
MITF- melanomas Tissue section Cell line
Pt#95
Pt#5
Hoek, K. S. et al. In vivo switching of human melanoma cells between proliferative and invasive states. Cancer Res. 68, 650–656 (2008).
Sensi M. et al. Human cutaneous melanomas lacking MITF and melanocyte differentiation antigens express a functional Axl receptor kinase. J Invest Dermatol. 131:2448-57, 2011.
-
2010
Background 2. NFAT family of Transcription Factors in normal and neoplastic cells
-
NFATc2 NFATc11
10
100
MF
INFATc2 NFATc1
50m 50m
Me#29318
BRAFV600E
p53WT
Me#15392
BRAFV600E
p53WT
Me#3046/2
BRAFV600E
p53n.d.
NFATc1
NFATc2
N N C C N N C C N N C C
+ - + - + - + - + - + - FBS
PARP
Tubulin
120 kDa
55 kDa
140 kDa
89 kDa
Perotti et al. J. Invest. Dermatol. 2012
Background 3. NFATc2 is a potential therapeutic target in melanoma
No
rmal
ized
d
ensi
ty v
alu
e
-
Hypothesis: NFATc2 may be an intrinsic regulator of melanoma dedifferentiation
NFATc2 expression correlates with a melanoma dedifferentiated profile
+ -
Outline of main findings
NFATc2
MITF and MITF-regulated genes
+ -
CD271 + -
-
MART-1 18 kDa
NFATc2
42 kDa β-actin
MITF
120 kDa
56 kDa
52 kDa GPNMB
PGC-1α 90 kDa
NFATc2 120 kDa
55 kDa α-Tubulin
27 kDa Rab27a
MITF 56 kDa
52 kDa
125 kDa
75 kDa
Protein level: NFATc2 expression more frequent in melanomas lacking MITF and proteins coded for by MITF target genes
NFATc2 is preferentially expressed in melanoma cells with a dedifferentiated profile
Inverse correlation between NFATc2 and Melanocyte Differentiation Antigens (MDA)
Perotti et al. Oncogene 2015
qPCR: Inverse correlation between NFATc2 and MITF
Protein level Inverse correlation between
NFATc2 and MITF in 24 cell lines
-
Inverse correlation between CD271 and Melanocyte Differentiation Antigen expression in melanoma cell lines
NFATc2 is preferentially expressed in melanoma cells with a dedifferentiated profile
MITF + -
+ - NFATc2
42k Da
120k Da NFATc2
CD271
β-actin
75k Da
CD271 expression in NFATc2+ cell lines
Quantitative assessment of western blot data
CD271 and NFATc2 expression by flow cytometry in 24 cell lines
-
Targeting of NFATc2 promotes melanoma differentiation
NFATc2
MART-1
GPNMB
120 kDa
56 kDa
52 kDa
95 kDa
18 kDa
75 kDa
125 kDa
90 kDa
42 kDa
MITF
gp100
PGC-1α
β- actin
MITF
Me71
75 kDa CD271
β-actin 42 kDa
Me71
siRNA
42 kDa
90 kDa
125 kDa
75 kDa
Me71
β-actin
PGC-1α
GPNMB
120 kDa
42 kDa
52 kDa
95 kDa
18 kDa
56 kDa
Me71
NFATc2
MART-1
β-actin
MITF
gp100
shRNA
β-actin
PGC-1α
GPNMB
42 kDa
90 kDa
125 kDa
75 kDa
Me71
NFATc2
MART-1
β-actin
MITF
gp100
120 kDa
42 kDa
52 kDa
95 kDa
18 kDa
56 kDa
Me71
NFATc2 inhibitor
Me71 Me71
Ctrl
shRNA_14
Ctrl
shRNA_15
NFATc2
shRNA_86a
NFATc2
shRNA_86b
NFATc2
shRNA_87a
Me71
NFATc2 shRNA_86a NFATc2 shRNA_87a Ctrl shRNA_15
gp100
-
NFATc2
MITF
GPNMB RAB27a PGC-1a Gp100 Tyrosinase MART-1 Melanin production Pigmentation
- - - - - - - - + + + + + + + +
+ CD271 -
Targeting of NFATc2 rescues melanoma differentiation
Genes modulated by NFATc2 silencing
Gen
es m
od
ula
ted
b
y TN
Fa
Up Down
Up
Down
46 49
41 14
TNF-a
T cells
TNF-a
TNF-a
TNF-RI/II
-
NFATc2+ melanomas express surface-bound TNF-a
NFATc2 profile
+ + + + + + - - -
mTNF-α
Events
TNFR1 TNFR2 TNF-α
NFATc2+ cell line (Me71)
Surface
staining Intracellular
staining
Surface
staining Surface
staining
Events
mTNF-α TNFR1 TNFR2 TNF-α
NFATc2- cell line (Me45)
Surface
staining Intracellular
staining
Surface
staining
Surface
staining
mRNA expression Protein expression
mTNFa correlates with TNFR1/2 NFATc2 correlates with TNFR1/2
-
Targeting of NFATc2 suppresses endogenous TNF-a
Ctrl siRNA
NFATc2
siRNA-1
mTNF-α
Ctrl siRNA
NFATc2
siRNA-2
Events
TNF-α
Events
Surface staining intracellular staining
Ctrl siRNA
NFATc2
siRNA-1
Ctrl siRNA
NFATc2
siRNA-2 Me71
Events
TNF-α
Events
Ctrl
shRNA_15
NFATc2
shRNA_86a
mTNF-α
Me71
Ctrl
shRNA_15
NFATc2
shRNA_87a
Ctrl
shRNA_15
NFATc2
shRNA_86a
Ctrl
shRNA_15
NFATc2
shRNA_87a
Endogenous TNF-a is under NFATc2 control
MITF
Ctrl 0.06 0.12
β-actin 42 kDa
MART-1 18 kDa
56k Da
52 kDa
Ab anti-TNF-α
(μg/ml)
NFATc2+ MITFlo mTNF-a+
cell line
MART-1
- 0.06 0.12 - 0.06 0.12
Ab anti-TNF-α
(μg/ml)
Ab anti-TNF-α
(μg/ml)
rTNF-α
42 kDa
18 kDa
β-actin
- - - + + +
Me71
rTNF-α
75 kDa CD271
β-actin 42 kDa
Me71
Role of endogenous TNF-a in melanoma dedifferentiation
-
NFATc2-TNFa axis
NFATc2
MITF
TNF-a
TNF-a
TNF-RI/II
?
C-Myc Brn2
Upstream regulator analysis of genes modulated by NFATc2 silencing
-
NFATc2
expression
Brn2 45 kDa
56 kDa
52 kDa MITF
Vinculin 110 kDa
-/+ + ++ +/- - - + + + ++ - +
45 kDa Brn2
56 kDa
52 kDa MITF
110 kDa Vinculin
45 kDa Brn2
56 kDa
52 kDa MITF
110 kDa Vinculin
Me71 Me98
Preferential expression of Brn2 in MITF- NFATc2+ melanomas
Targeting of Brn2 upregulates MITF
-
c-myc 65 kDa
57 kDa
42 kDa β-actin
120 kDa NFATc2
Me71
45 kDa Brn2
c-myc 65 kDa
57 kDa
42 kDa β-actin
120 kDa NFATc2
Me98
Brn2 45 kDa
65 kDa
57 kDa c-myc
42 kDa β-actin
120 kDa NFATc2
Me71
45 kDa Brn2
120 kDa NFATc2
42 kDa β-actin
65 kDa
57 kDa c-myc
Me71
45 kDa Brn2
Targeting of NFATc2 suppresses c-Myc and Brn2
MART-1 18 kDa
β-actin 42 kDa
MITF 56k Da
52 kDa
Ctrl 0.06 0.12
Ab anti-TNF-α
(μg/ml)
65 kDa
57 kDa c-myc
Me71 Me98
65 kDa
57 kDa c-myc
45 kDa Brn2
56 kDa
52 kDa MITF
β-actin 42 kDa
rTNF-α
MART-1 18 kDa
NFATc2 120 kDa
MITF 56 kDa
52 kDa
β-actin 42 kDa
65 kDa
57kDa c-myc
42 kDa β-actin
45 kDa Brn2
56 kDa
52 kDa MITF
65 kDa
57 kDa c-myc
Me71
Endogenous TNF-a regulates c-Myc and Brn2
NFATc2 TNF-a c-Myc Brn2 MITF
-
Targeting of c-Myc suppresses Brn2 and rescues MITF expression
65 kDa
57 kDa c-myc
Brn2 45 kDa
56 kDa
52 kDa MITF
18 kDa MART-1
42 kDa β- actin
c-myc 65 kDa
57 kDa
56 kDa
52 kDa MITF
45 kDa Brn2
18 kDa MART-1
42 kDa β- actin
Me71 Me98
18 kDa MART-1
c-myc 65 kDa
57 kDa
45 kDa Brn2
56 kDa
52 kDa MITF
42 kDa β- actin
65 kDa
57 kDa
c-myc
45 kDa Brn2
56 kDa
52 kDa MITF
42 kDa β-actin
18 kDa MART-1
Me71 Me98
NFATc2 TNF-a c-Myc Brn2 MITF
-
NFATc2 TNF-a
TNF-a
TNF-RI/II
Brn2 C-Myc MITF
GPNMB RAB27a PGC-1a Gp100 Tyrosinase MART-1 Melanin production Pigmentation T cell recognition CD271
- - - - - -
+
- - -
GPNMB RAB27a PGC-1a Gp100 Tyrosinase MART-1 Melanin production Pigmentation T cell recognition CD271 -
+ + + + + + + + +
Oncogene, 2015
The final model
-
NFATc2 and MITF expression HLA-A2, b2M expression and
CTL-mediated lysis MDA expression
NFATc2HI MITFLo melanoma cell lines are poorly recognized by MDA-specific CTLs
-
CTL lines to
MART-127-35
MART-127-35
gp100209-217
*** *** ***
*** ***
*** *** ***
*** ***
***
*** *** ***
**
*
***
***
* **
Ctrl shRNA_15
NFATc2 shRNA_86a
NFATc2 shRNA_87a
Ctrl shRNA_15
NFATc2 shRNA_86a
NFATc2 shRNA_87a
Ctrl shRNA_15
NFATc2 shRNA_86a
NFATc2 shRNA_87a
Targeting of NFATc2 improves CTL-mediated melanoma recognition
a
Eve
nts
HLA-A2
Ctrl
shRNA_15
NFATc2
shRNA_86a
Ctrl
shRNA_15
NFATc2
shRNA_87a
CTL lines to
MART-127-35
MART-127-35
Gp100209-217
b
Me 71 Ctrl shRNA_15
Me 71 Ctrl shRNA_15
Me 71 Ctrl shRNA_15
-
NFATc2
NFATc2 vs MITF in-vivo
MITF NFATc2
-
Conclusions
Acknowledgements
Human Tumors Immunobiology Unit
Roberta Mortarini Valentina Perotti Elena Tassi Giulia Grazia
Paola Baldassari Ilaria Bersani Alessandra Molla Claudia Vegetti Gabriella Nicolini
Surgical Oncology Unit Andrea Maurichi Mario Santinami
Dept. of Pathology Gabrina Tragni
Functional Genomic Core Facility Loris de Cecco Edo Marchesi Silvana Canevari
Grant support
-Expression of NFATc2 promotes a MITF- CD271+ dedifferentiated profile in melanoma cells. -NFATc2 promotes expression of mTNF-a that in turn negatively regulates MITF and differentiation through a Myc-Brn2 axis. -Melanoma differentiation can be rescued by targeting NFATc2, Myc and Brn2 and by neutralization of endogenous TNF-a. -Targeting of NFATc2 improves recognition of melanoma cells by MDA-specific CTLs.
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