Download Development of differentiation assay in neuroblastoma to elucidate

Development of differentiation assay in neuroblastoma
to elucidate the role of MYCN in differentiation
Zuzanna Urban1, Evon Poon1, Louise Howell1, Kevin Petrie1, Louis Chesler1,2
Background
Neuroblastoma is the most common extracranial solid tumour in children; patients with undifferentiated neuroblastoma subtypes have a poor prognosis with 5-year
overall survival of only 50%. Amplification of MYCN and overexpression of its coded protein is associated with rapid tumour progression and poor outcome.
Induction of terminal differentiation is a very promising approach to neuroblastoma treatment. However, there’s no golden standard to asses neuroblastoma
differentiation and therefore the aim of this project was to develop such assay. Using a well-established differentiation agent, retinoic acid, we developed a reliable
differentiation assay by combining analysis of neurite outgrowth and expression of numerous neuronal markers.
MYCN plays an important role in stem cell homeostasis and cell proliferation. Several contradictory studies have shown that MYCN either decreases upon retinoic
acid treatment (Amatruda, Sidell et al. 1985, Thiele, Reynolds et al. 1985) or is necessary for the onset of differentiation (Guglielmi, Cinnella et al. 2014). We used
our differentiation assay to study the role of MYCN in differentiation.
Results
BE2C
DAPI
MYCN
b)
NF-L
K e lly
BE2C
100
10
*
c o n tr o l
1  M A tR A
5
*
C R AB P 2
c)
R AR 
T rkB
1  M A tR A
****
40
20
**
R AR 
TRKB
RET
NF-L
siMYCN
NF-L
control
MYCN
MYCN
Kelly
BE2C
Kelly
DAPI
BE2C
DAPI
****
60
C R AB P 2
Ret
a)
c o n tr o l
80
0
0
AtRA
**
*
R e la tiv e e x p re s s io n
R e la tiv e e x p re s s io n
control
*
scrambled
a)
Lan5
Tet21
MYCN
NF-L
DAPI
MYCN
NF-L
control
control
DAPI
Tet21 DOX
b)
NF-L
AtRA
AtRA
actin
DOX
AtRA
-
+
+
-
c)
+
+
NFL
GAPDH
R e la tiv e e x p re s s io n
c o n tro l
C-MYC
A tR A
0 .5
0 .0
T e t2 1
2 .5
**
s iM Y C N
2 .0
1 .5
1 .0
0 .5
B E 2C
M Y C N m R N A e x p r e s s io n
1 .0
R e t e x p r e s s io n
0 .0
1 .5
MYCN
Lan5
c)
si Scr
Scr
MYCN
b)
BE2C
si Scr
R e la tiv e e x p re s s io n
a)
NF-L
siMYCN
AtRA
Figure 1. Neuroblastoma cells differentiate upon treatment with alltrans retinoic acid (AtRA); a) 14-days treatment with AtRA induces
neurite outgrowth in Kelly and BE2C cells, overall MYCN levels are
not affected but cells with neurites have low MYCN expression
(arrows); b) AtRA treatment induces expression of RA-response
genes (CRABP2 and RARβ) and neuronal differentiation genes
(TrkB, Ret); c) Transmission electron microscopy showing
neurosecretory granules in BE2C and Kelly cells treated with AtRA.
MYCN
scrambled
DAPI
Figure 2. AtRA treatment leads to decrease in MYCN
expression in SHEPTet21 (cell line with doxocyclineinducible MYCN expression); a) immunofluorescent
staining of Tet21 cells expressing MYCN (left panel) and
not expressing MYCN (right panel); b) whole-protein
lysates of Tet21 cells; c) MYCN mRNA expression in Tet21
cells measured by qPCR.
Lan5
Figure 3. MYCN silencing leads to differentiation of
neuroblastoma cell lines; a) transfection of BE2C and Lan5 cell
lines with MYCN-directed siRNA; cells with longest neurites have
low levels of MYCN (arrows); picture taken after 72h b) Western
blot analysis confirms partial knock-down of MYCN and increase
in NF-L expression; c) MYCN knock down leads to upregulation
of Ret (neuronal differentiation marker).
T e t2 1 D O X
Conclusions
Poorly differentiated cancers present an aggressive phenotype – they grow quickly, have higher metastatic potential and are correlated with unfavourable outcome.
The idea of differentiation therapy is therefore well understood. However, there’s no standard method to assess differentiation in neuroblastoma cells. Therefore we
developed a reliable and robust differentiation assay to correctly distinguish differentiated cells i.e. after treatment with a drug.
We used the assay to establish a correlation between differentiation and MYCN expression. AtRA (a well known differentiation agent) leads to increase in
neurofilament-light expression; we observed that cells with particularly long neurites have low MYCN expression. To study it further, we used a MYCN-inducible
SHEP-Tet21 cell line in which treatment with AtRA lead to clear downregulation of MYCN protein and mRNA expression. Knock-down of MYCN in MYCN-amplified
cell lines was not entirely efficient but again, cells with particularly long neurites had low MYCN expression.
All these results support the conclusion that MYCN inversely correlates with differentiation, which has been suggested before but has never been shown so clearly.
Contact
Acknowledgments
[email protected]
FUNDING ACKNOWLEDGEMENT
This project has received funding from the European Union’s
Seventh Framework Programme for research, technological
development and demonstration under grant agreement no
315902. Zuzanna Urban gratefully acknowledges receipt of a Marie
Curie Research Fellowship. Kevin Petrie and Louis Chesler are
Partners within the Marie Curie Initial Training Network DECIDE
(Decision-making within cells and differentiation entity therapies).
1
Institute of Cancer Research, 15 Cotswold Road, SM2
5NG, Sutton, Surrey
2 The Royal Marsden NHS Trust, Downs Road, SM2 5PT
Sutton, Surrey, UK
We thank Dr Lucy Collinson and Dr Anne Weston from The
Francis Crick Institute in London for all the help with electron
microscopy.