Transcript The DREAM Complex Mediates GIST Cell Quiescence and Is a

The DREAM Complex Mediates GIST
Cell Quiescence and Is a Novel
Therapeutic Target to Enhance
Imatinib-Induced Apoptosis
Sergei Boichuk, Joshua A. Parry, Kathleen R. Makielski,
et al. 2013; 73:5120-5129.
Published OnlineFirst June 20, 2013.
-Cancer Reasearch
报告人：焦鑫艳
报告时间：2014.9.17
Cancer
Research
(Cancer Res) 杂志，为
美国癌症研究协会
(American Association
for Cancer Research，
AACR) 会 刊 。 1916 年
创 刊 ， 半 月 刊 ，
Pubmed和SCI收录。
主要发表包括基础研究、
临床前及临床、肿瘤预
防及生物治疗在内的肿
瘤学原创研究论文和综
述文章，为国际肿瘤研
究领域引用率最高的杂
志之一。
1999-2012
目前中国
人在该杂
志累计发
表343篇。
2001-2012
近四年的影响因子：
2010
2011
2012
2013
8.234
7.856
8.65
9.284
Background
1. The majority of gastrointestinal stromal tumors (GIST), the most common
mesenchymal tumor of the gastrointestinal tract , are characterized by oncogenic
mutations in the KIT or platelet-derived growth factor receptor-a (PDGFRA)
receptor tyrosine kinase.
2. GIST can be successfully treated with the small-molecule kinase inhibitor
imatinib mesylate (Gleevec). Complete remissions are rare and patients frequently
achieve disease stabilization in the presence of residual tumor masses.
3. Discontinuation of treatment can lead to tumor progression. Residual tumor
cells are quiescent， remain viable and able to re-enter the cell-division cycle.
4. This reversible exit from the cell division cycle and entry into G0 has
previously been shown to involve the anaphase-promoting complex the APCCDH1 –
SKP2–p27 Kip1 signaling axis.
APC, together with its activator CDH1, promotes the polyubiquitylation and
subsequent degradation of SKP2, a substrate adaptor component of the SCF
(SKP1–Cullin–F-box) complex. SKP2 loss results in the accumulation of its target,
the CDK inhibitor p27 Kip1, and the reinforcement of a quiescent state.
In previous study, we could show that this process is active in imatinib-treated
GIST cells.
5. A second major group of proteins that negatively regulate the cell cycle.
p130 has been shown to accumulate in G0 and is regulated by SKP2.
p130 interacts with E2F4 to repress E2F-dependent gene transcription.
This DREAM complex is a multisubunit protein complex in mammalian， consists
of DP, RBL2 (p130), E2F4 and the mammalian homologs of the Caenorhabditis
elegans (C. elegans) synthetic multivulva class B (synMuvB) core gene products
LIN9, LIN37, LIN52, LIN53/RBBP4, and LIN54.
Specificity tyrosine-phosphorylation–regulated kinase (DYRK), DYRK1A,
mediated pLIN52-Ser28 of the DREAM component and pLIN52-Ser28 was shown
to regulate complex formation in G0.
6. Imatinib induces GIST cell quiescence in vitro through the APCCDH1 –SKP2–
p27 Kip1 signaling axis.
7. DREAM complex, a multisubunit complex that has recently been identified as
an additional key regulator of quiescence.
8. Here, we provide evidence that imatinib induces GIST cell quiescence in vivo and
that this process also involves the DREAM complex.
Material
The human GIST cell line GIST882
(derived from an untreated metastatic GIST)
For mouse xenograft models
GIST882 cells [carrying a KIT p.K642E (exon 13) mutation)] or tumors
originating from the biopsies of two patients [bearing KIT p.V650D (exon 11) or
KIT p.A502_Y503dup (exon 9) mutations, respectively] were implanted in both
flanks of two mice.
Second-passage xenografts were generated by explanting established
xenografts and implanting them into the flanks of a second set of mice.
Methods
1. Cell culture, inhibitor treatments, and siRNA-transfections
2. Immunologic and cell-staining methods
( Coimmunoprecipitation and immunofluorescence analysis)
3. BrdUrd assay
4. Senescence-associated β-galactosidase activity (~ staining kit)
5. Quantitative real-time reverse transcriptase PCR
6. Cell-cycle analysis (Flow Cytometry)
7. GIST xenograft models
Sections
1. Imatinib induces GIST cell quiescence in vivo and in vitro.
Fig1. 2 S1-S3
2. The DREAM complex is involved in imatinib-induced quiescence.
Fig3
3. The DREAM complex is a modulator of the cellular response to imatinib and is
a potential therapeutic target.
Fig4. 5 S4
Immunofluorescence microscopic analysis
Figure 1.
Imatinib induces GIST cell quiescence in vivo.
the quiescence marker p27 Kip1
mutation
Immunohistochemical analysis
Figure S1.
Expression levels of its upstream regulator
SKP2 do not predict p27Kip1 levels after
imatinib therapy.
BrdUrd incorporation
Cellular proliferation detection of
the percentage of cells in S-phase
Figure 1.
Imatinib induces GIST cell quiescence in vitro.
细 DNA合成前期（G1期）
细 胞
胞 间 DNA合成期（S期）
分 期 DNA合成后期（G2期）
裂
M期为细胞分裂期
G0期：离开细胞周期，
停止细胞分裂。
A subset of GIST cells
showing
morphologic
signs of apoptosis during
imatinib treatment and
cell growth completely
recovers after imatinib
washout.
Figure S2.
Imatinib treatment leads
to quiescence and not
senescence in GIST cells
Immunoblot analysis
Figure 2.
Imatinib-induced GIST cell quiescence does
not prevent apoptosis upon imatinib rechallenge.
Detection of KIT activation and markers of
cell-cycle regulation, markers of apoptosis.
Cell-cycle reentry after removal of imatinib
Pretreated GIST cells retain their
esponsiveness to antineoplastic activities of
imatinib as suggested by previous clinical
reports.
No change in the percentage of
senescence-associated
β–
galactosidase (SA β–gal) positive
cells
B
Senescence-associated marker
p16 INK4A (CDKN2A)
Immunofluorescence microscopic staining
Figure S3.
Imatinib does not induce a
senescence phenotype in these
cells.
DREAM complex
members: p130
(accumulate in G0),
E2F4, and LIN37
Immunofluorescence microscopic analyses
Figure 3.
The DREAM complex
is
involved
in
imatinib-induced
quiescence.
Enhanced formation
of a complex among
p130,
E2F4,
and
LIN37 after imatinib
treatment of GIST
cells.
Figure S4.
Knockdown of single DREAM
complex subunits (p130, E2F4,
LIN9, LIN37, or LIN54) did not
result in a significant increase of
imatinib-induced
GIST
cell
apoptosis
E2F4/LIN54
knockdown
enhanced GIST cell apoptosis.
Inhibition of efficient DREAM
complex formation resulted in
increased baseline proliferation.
Figure 4.
LIN52 is activated by imatinib
and attenuates its proapoptotic
activities.
pLIN52-S28 increased. Basal
expression of DYRK1A and
LIN52 remained unchanged.
p-LIN52 and DREAM complex
formation were reversible after
removal of imatinib.
A cell-cycle arrest in G2–M in
LIN52-depleted GIST cells. A
statistically significant increase
of apoptosis.
Figure 5.
Inhibition
of
DYRK1A
enhances
imatinib-induced
GIST cell apoptosis.
DYRK1A is a protein kinase.
DYRK1A inhibitor harmine
markers of apoptosis (PARP
cleavage, cleaved caspase 3)
and quiescence (p130)
DAPI
stain
morphologically
apoptotic cells
to
detect
Here, we show that
1.Imatinib induces GIST cell quiescence in vivo and in vitro.
2. This process involves the DREAM complex as evidenced by upregulation of
p130, increased p130/E2F4/LIN37 complex formation, and enhanced
phosphorylation of the DREAM subunit LIN52.
3. Importantly, inhibition of DREAM complex formation, abrogation of
quiescence by siRNA-mediated knock-down of LIN52 or the DYRK1A kinase
were both found to significantly increase imatinib-induced GIST cell apoptosis.
4. Therefore, interference with DREAM-mediated quiescence can enhance
imatinib-induced apoptosis and anti-GIST cell activity, which emphasizes the
relevance of the DREAM complex as novel drug target for more efficient imatinib
responses.
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