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Harnessing the power of stem cell self-renewal pathways in cancer: dissecting the role of BMI-1 in Ewing’s sarcoma initiation and maintenance
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Harnessing the power of stem cell self-renewal pathways in cancer: dissecting the role of BMI-1 in Ewing’s sarcoma initiation and maintenance
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Content
HARNESSING THE POWER OF STEM CELL SELF-RENEWAL PATHWAYS IN
CANCER: DISSECTING THE ROLE OF BMI-1 IN EWING’S SARCOMA
INITIATION AND MAINTENANCE
by
Hao-Ru Jessie Hsu
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR, AND CELLULAR BIOLOGY)
August 2010
Copyright 2010 Hao-Ru Jessie Hsu
ii
Acknowledgements
I owe my deepest gratitude to my Ph.D. thesis advisor, Dr. Elizabeth
Lawlor. This work would not have been possible without her support,
encouragement, and guidance. Throughout my career as a Ph.D. student, she
has been with me ups and downs, both professionally and personally. She is
both a mentor and a friend, looking out for the best interest of my career and life.
She taught me the basics of being a scientist and a human being. Her scientific
ideas are exciting and refreshing. I had the greatest pleasure working with her.
I am indebted to members of the Lawlor Lab family (not in any particular
order), Long Hung, Aaron Cooper, Ynnez Gwye, Chris Scannell, Darren Russell
and Drs. Cynthia Jiang, Dorothea Douglas, John van Doorninck, Cornelia von
Levetzow, Gregor von Levetzow, for all the support they have given me.
Together, they made the Lawlor Lab a fun and exciting place to work and play. It
was their teamwork and effervescent attitude that made all the hardship of
scientific discovery seemed less painful. I especially want to thank Dr. Dorothea
Douglas, who contributed greatly to the conception of our BMI-1 project. Dr.
Douglas also performed soft agar assays and helped with in vivo xenograft
experiments that were essential in identifying the BMI-1-mediated phenotype in
ESFT. I would like to acknowledge Aaron Cooper for all his efforts on cloning our
constitutive and inducible EWS-FLI1 lenti-viral vectors, as well assistance on
many aspects of my thesis project. Finally, I would like to thank Long Hung for
providing training and assistance on innumerable aspects of my projects and life,
iii
not limited to cloning vectors, derivation of cell lines, adhesion assays, bringing
food re-enforcement, and qRT-PCR, and FACS analysis. I would not have been
able to juggle multiple experiments at the same time without his assistance.
I am grateful to members of the Triche Lab, Daniel Wai, Sheetal Bajaj,
John Kaddis and Drs. Diana Abdueva and Michele Wing for various technical
and emotional support. The entire experience of getting a Ph.D. would not have
been the same without their stimulating company. In particular, I would like to
thank Dr. Diana Abdueva for processing and analyzing microarray data related to
my BMI-1 studies. I would also like to thank Daniel Wai for sharing his WNT5A
conditioned medium and all his WNT signaling-related reagents.
I am thankful to our collaborators, Drs. Heinrich Kovar, Carolyn Lutzko,
Gerard Evan, Timothy Triche, and Peter Laird for cells and reagents. I would like
to acknowledge the CHLA genomic, animal, vector, and FACS core for various
technical assistance. I would like to acknowledge Dr. Peter Laird and his team for
helping us with DNA methylation studies using the Illumina GoldenGate platform.
The faculties and staffs of the PIBBS/GMCB program have greatly helped
facilitate my Ph.D. study. I am grateful everyday for the opportunity to pursue a
doctoral degree here at USC. I would like to acknowledge the USC California
Institute for Regenerative Medicine (CIRM) training grant for financially
supporting me during the last two years of my Ph.D study. I especially like to
thank members of my Ph.D. thesis committee, Drs. Debbie Johnson, Baruch
Frenkel, and Mike Stallcup for all their helpful advise and stimulating discussion.
iv
To my parents, whose unconditional support and love gave me courage
and independence to create and discover my own path in life. I am forever
indebted and grateful to them.
Finally, I am grateful to all the ESFT patients for sharing with us molecular
samples of their disease, and the donors who generously provided financial
support for our research. Although I am greatly intrigued by the pathogenesis of
ESFT, I do wish that all ESFT patients could be disease free one day. I am
optimistic that with the collaboration of scientists and clinicians, we will
successfully develop therapeutic strategies to fully eradicate this aggressive
disease in the future.
v
Table of Contents
Acknowledgements ii
List of Figures vi
Abbreviations viii
Abstract ix
Chapter 1: Background and significance 1
Chapter 2: BMI-1 functions as an oncogene in Ewing’s sarcoma 5
Chapter 2 Introduction 5
Chapter 2 Results 8
Table 1: Gene ontology biological processes 25
significantly affected by BMI-1 knockdown
in A4573 ESFT cells.
Chapter 2 Discussion 37
Chapter 3: BMI-1 cooperates with YAP to suppress cell contact 45
inhibition in Ewing’s sarcoma
Chapter 3 Introduction 45
Chapter 3 Results 48
Chapter 3 Discussion 63
Chapter 4: EWS-FLI1 and BMI-1 cooperate in Ewing’s sarcoma 69
tumorigenesis
Chapter 4 Introduction 69
Chapter 4 Results 74
Chapter 4 Discussion 91
Chapter 5: From bench to bedside: a new hope for BMI-1-driven 96
ESFT therapies
Chapter 6: Material and methods 99
Bibliography 109
Appendix: Peer-reviewed publication in Cancer Research (2008) 124
BMI-1 promotes Ewing sarcoma tumorigenicity independent
of CDKN2A repression.
vi
List of Figures
Figure 1: BMI-1 represses the CDKN2A locus 4
Figure 2: Ewing tumors (ESFT) express BMI-1 10
Figure 3: BMI-1 knockdown does not affect ESFT cell proliferation 12
or death
Figure 4: CDKN2A/p16 status of ESFT cell lines 15
Figure 5: BMI-1 modulation does not affect CDKN2A levels 16
or p16-RB pathway
Figure 6: BMI-1 promotes the anchorage-independent growth 20
of ESFT cells
Figure 7: BMI-1 promotes ESFT tumorigenicity in vivo 22
Figure 8: Gene expression profiling of A4573 BMI-1 knockdown 24
and control cells
Figure 9: Comparison of gene expression pattern between 27
BMI-1 knockdown ESFT and medulloblastoma cells identified
101 overlapping BMI-1 targets
Figure 10: BMI-1 accelerates ESFT cell adhesion in vitro 29
Figure 11: BMI-1 alters the adhesion property of ESFT 33
through WNT5A repression
Figure 12: Hyper-methylation of NID1 and WNT5A promoters 36
in ESFT correlates with BMI-1 expression
Figure 13: Knockdown of BMI-1 in ESFT restores contact inhibition 49
Figure 14: Loss of BMI-1 induces death and cell cycle arrest 53
cell-contact inhibition
Figure 15: Loss of BMI-1 destabilizes YAP oncoprotein, 58
a downstream effector of cell-contact inhibition
Figure 16: YAP functions as an oncogene in ESFT 61
vii
Figure 17: Ectopic EWS-FLI1 expression induces growth arrest 75
in MRC5 primary human fibroblasts
Figure 18: BMI-1 abrogates EWS-FLI1-induced growth arrest 77
in MRC5 cells
Figure 19: BMI-1 abrogates EWS-FLI1-induced oncogene- 79
induced senescence in MRC5 cells
Figure 20: EWS-FLI1 expression is maintained one-month 81
post-transduction
Figure 21: Development of a doxycycline-inducible EWS-FLI1 84
expression system in MRC5 cells
Figure 22: Possible mechanism of BMI-1-mediated protection 86
against EWS-FLI1-induced OIS
Figure 23: EWS-FLI1 does not regulate BMI-1 in ESFT cells 88
Figure 24: hTERT-immortalized MRC5 cells tolerate EWS-FLI1 90
viii
Abbreviations
AKT v-akt murine thymoma viral oncogene
BMI-1 B lymphoma Mo-MLV insertion region 1
BSA Bovine serum albumin
DNMT DNA methyltransferase
ESFT Ewing’s sarcoma family of tumor
EGFP Enhanced green fluorescence protein
FBS Fetal bovine serum
hESC Human embryonic stem cell
hTert Human telomerase reverse transcriptase
HRP Horseradish peroxidase
Hygro Hygromycin B
MSC Mesenchymal stem cell
MUT Mutant
NID1 Nidogen 1
OIS Oncogene-induced senescence
Puro Puromycin
PVDF Polyvinylidene fluoride
QRT-PCR Quantitative real-time polymerase chain reaction
RB Retinoblastoma suspectibility protein
RT-PCR Reverse transcription polymerase chain reaction
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
TBST Tris-buffered saline with tween-20
WNT5A Wingless-type MMTV integration site family, member 5A
WT Wild-type
YAP Yes-associated protein
5Aza-dC 5-aza-2’ deoxycytidine
ix
Abstract
The stem cell self-renewal pathway is often hijacked and over-activated in
cancer cells. The polycomb group protein BMI-1 is a known regulator of
stemness and its over-expression is associated with a number of malignancies.
In this study, we have examined the role of BMI-1 in the pathogenesis of Ewing’s
sarcoma family of tumors (ESFT). We hypothesize that deregulation of BMI-1 –
driven signaling pathway is central to the initiation and maintenance of this family
group of aggressive pediatric tumor. To assess our hypothesis, we have
performed gain and loss of function studies and provided evidence to support the
role of BMI-1 in promoting anchorage-independent growth of ESFT in vitro and
tumorigenicity in vivo independent of the genetic status of the cells. Contrary to
early indications in stem cells and other caner cell types, BMI-1 promotes growth
of ESFT cells independent of CDKN2A repression. To identify novel BMI-1
downstream targets involved in BMI-1 mediated tumorigenicity, we have
conducted gene expression profiling on ESFT cells following BMI-1 knockdown.
Significantly, we discover hundreds of putative BMI-1 targets including genes
involved in cell adhesion, development and differentiation. In particular, genes
associated with cell-cell and cell-matrix adhesion are over-represented. In light of
this data, we have focused our investigation on the role of BMI-1 in cell adhesion.
Using in vitro adhesion assays, we find that BMI-1 accelerates ESFT adhesion in
part through repression of the basement protein Nidogen 1 and a member of the
WNT protein family, WNT5A. These studies indicate that BMI-1 may promote
x
tumor growth and engraftment through modulation of adhesion pathways and
non-canonical WNT signaling.
To further dissect the role of BMI-1 in influencing ESFT phenotype, we
investigated the potential contribution of BMI-1 to other hallmarks of cancer, in
particular, loss of cell contact inhibition, a fundamental transforming property of
cancer cells. Using cells that stably express an shRNA against BMI-1, we have
discovered that loss of BMI-1 expression restores contact inhibition to ESFT
cells. Significantly, although proliferation of cells in log phase growth is
unaffected by loss of BMI-1, at high cell density BMI-1 knockdown cells undergo
cell cycle arrest and death. In contrast, control vector-transduced cells continue
to enter cell cycle and proliferate, avoiding contact inhibition. Although many
signaling pathways are involved in mediating the cell contact inhibition response,
inactivation of the Yes-Associated Protein (YAP), a key downstream target of the
Hippo pathway, has been implicated in both Drosophila and mammalian cells.
Intriguingly, our data show that loss of BMI-1 expression results in loss of YAP
protein at high cell density coincident with induction of contact inhibition. Further,
silencing of YAP markedly decreases the tumorigenic potential of ESFT cells,
implicating its role as a cooperative oncogene in ESFT. Together, our finding
suggests that YAP is a novel downstream target of BMI-1 and that BMI-1-
mediated stabilization of YAP renders cancer cells impervious to cell contact
inhibition. Cancer cells escape cell contact inhibition to achieve unrestrained
proliferation and enhanced invasive and metastatic properties. Our findings
xi
suggest that BMI-1 may be a key upstream mediator of this critical cancer-
associated phenotype.
Finally, the cell of origin of ESFT remains elusive. We hypothesize that
high levels of BMI-1 expression is an inherent feature of its cell of origin and its
activity is required in cooperation with EWS-FLI1, a characteristic molecular
feature of the majority of ESFT cells, for ESFT initiation. To this end, we have
investigated the role of BMI-1 in EWS-FLI1-driven malignant transformation in
primary human fibroblasts. Studies by Lessnick et al. have shown that EWS-FLI1
is toxic to primary human fibroblasts in a p53-dependent manner. Intriguingly, our
study shows that co-expression of BMI-1 and EWS-FLI1 in primary human
fibroblasts renders cells tolerant for EWS-FLI1 expression. Importantly, we find
that EWS-FLI1 induces a senescent phenotype in MRC5 cells in the absence of
BMI-1 over-expression, as evidenced by positive staining for senescence-
associated-β-galactosidase activity. Together, these data suggests that BMI-1
abrogates EWS-FLI1-induced oncogene-induced senescence (OIS). Studies are
currently ongoing to dissect pathways involved in BMI-1-mediated protection
against EWS-FLI1-induced OIS.
The origin of elevated BMI-1 expression in ESFT is a mystery. Although
BMI-1 may be an inherent feature of the ESFT cell of origin, its expression may
also be induced by other ESFT-specific oncogenes such as EWS-FLI1. However,
our data show no consistent modulation of BMI-1 expression by EWS-FLI1 in
xii
primary human fibroblasts and ESFT cells. Future studies will be necessary to
decipher mechanisms underlying BMI-1 regulation.
In an attempt to identify other cooperative oncogenes of EWS-FLI1 in
addition to BMI-1 in primary human cells, we have found that hTERT-
immortalized primary human fibroblasts tolerated EWS-FLI1 expression just as
well as BMI-1-over-expressing primary human cells. This implicates a role of
hTERT in EWS-FLI1-mediated malignant transformation. It will be interesting to
determine if co-expression of BMI-1, hTERT, and EWS-FLI1 together will be
sufficient to reprogram primary human fibroblasts into malignant ESFT-like cells.
1
Chapter 1
Background and significance
Ewing’s sarcoma family of tumors (ESFT) is a group of highly aggressive
pediatric bone and soft tissue malignancies. Despite multimodal treatment
regimens, the survival rate for patients remains 75% for patients with localized
disease and <20% for patients with metastases at diagnosis(Balamuth and
Womer; Ban et al., 2006). The development of novel therapeutic strategies
remains challenging as the molecular nature and the cellular origin of ESFT
remain elusive.
Over 85% of the ESFT cases are characterized by t(11;22) chromosomal
translocation, resulting in the expression the chimeric EWS-FLI1 fusion protein.
EWS-FLI1 promotes the growth of ESFT and is widely believed to play a major
role in ESFT development and pathogenesis. Interestingly, not all primary human
cells tolerate EWS-FLI1 expression suggesting that not all cell types are
permissive for ESFT development. To date, primary human cells with inactivated
p53 tumor suppressor and human mesenchymal stem cells are the only cell
types that have been shown to tolerate EWS-FLI1 expression (Lessnick et al.,
2002; Riggi et al., 2008). However, it is noteworthy that the expression of EWS-
FLI1 in these aforementioned cells is not sufficient to induce malignant
transformation, suggesting that additional oncogenic mutations or epigenetic
changes are required in cooperation with EWS-FLI1 to render cells tumorigenic.
2
Stem cell self-renewal pathways are commonly deregulated in cancer
(Reya et al., 2001). The stem cell self-renewal factor BMI-1 is a member of the
polycomb group family of transcription repressors that are required for the self-
renewal of post-natal hematopoietic stem cells and neural stem cells(Molofsky et
al., 2005; Park et al., 2003). In addition to its role in the stem cell maintenance, it
also functions as an oncoprotein in a number of human malignancies, including
lymphoma (Bea et al., 2001; van Kemenade et al., 2001), leukemia (Chowdhury
et al., 2007; Sawa et al., 2005), glioma (Godlewski et al., 2008),
medulloblastoma(Leung et al., 2004; Wiederschain et al., 2007),
neuroblastoma(Cui et al., 2007; Nowak et al., 2006), non-small cell lung cancer
(Becker et al., 2009; Vonlanthen et al., 2001), breast cancer (Guo et al., 2007b;
Hoenerhoff et al., 2009; Liu et al., 2006b), prostate carcinoma (Berezovska et al.,
2006; Fan et al., 2008), and colon cancer (Kim et al., 2004). Intriguingly, BMI-1
has been reported to promote the self-renewal of a subpopulation of cancer cells
termed cancer stem cells in leukemia, breast cancer, brain tumor, and head and
neck cancer(Al-Hajj et al., 2003; Cui et al., 2006; Hemmati et al., 2003; Lessard
and Sauvageau, 2003; Liu et al., 2006b; Prince et al., 2007; Singh et al., 2003).
Moreover, studies on the mechanism underlying leukemia initiation illustrates that
BMI-1 cooperates with leukemic-associated fusion proteins TLS-ERG (an EWS-
FLI1 related fusion protein), E2A-PBX1, and HOXA9-MEIS1 to induce
leukemogenesis (Lessard and Sauvageau, 2003; Smith et al., 2003; Warner et
al., 2005). Together, these studies demonstrate that deregulation of stem cell
3
programs in association with BMI-1 plays an essential role in tumor initiation and
maintenance.
The molecular basis underlying BMI-1 mediated tumorigenicity and self-
renewal is dependent on the cellular context. In postnatal stem cells and some
cancer cells, BMI-1 promotes proliferation by repressing the CDKN2A locus,
which encodes two inhibitors of cyclin-dependent kinase, p16
INK4a
and ARF.
Repression of p16
INK4a
and ARF by BMI-1 leads to G1 cell cycle progression as a
result of Rb inactivation and abrogation of p53-mediated growth arrest,
respectively (Figure 1). In embryonic neural stem cells, Bmi-1 mediates their self-
renewal by repressing the Cdkn1a locus, which encodes a different cell cycle
inhibitor, p21/Cip1. In some cancer types, BMI-1 promotes tumorigenicity through
alternative CDKN1A and CDKN2A-independent mechanisms (Bruggeman et al.,
2007; Douglas et al., 2008; Wiederschain et al., 2007). Notably, pathways
involving integrin-mediated adhesion, DNA damage response, redox
homeostasis, and mitochondrial function have all been implicated downstream of
BMI-1 signaling (Bruggeman et al., 2009; Liu et al., 2009).
Our lab hypothesizes that BMI-1 deregulation is central to the
pathogeness of ESFT. In this thesis, I will delineate the role of BMI-1 in ESFT
maintenance and unravel the molecular mechanism underlying BMI-1 mediated
tumorigenicity. Further, the cooperative role of BMI-1 in EWS-FLI1-mediated
cellular transformation will be investigated to gain further insight on the initiation
of ESFT in primary human cells.
4
Figure 1. BMI-1 represses the CDKN2A locus. CDKN2A encodes cell cycle inhibitors, p16
INK4A
and ARF. p16
INK4A
prevents hyper-phosphorylation or inactivation of Rb tumor suppressor protein
by CDK4/6 • Cyclin D complex, thereby inhibiting G1 cell cycle progression. ARF promotes p53-
dependent activation of pro-apoptotic genes (e.g. p21) through suppressing the function of
MDM2. Thus, repression of the CDKN2A locus by BMI-1 leads to G1 cell cycle progression
5
Chapter 2
BMI-1 functions as an oncogene in Ewing’s sarcoma
Chapter 2 Introduction
Members of the Ewing sarcoma family of tumors (ESFT) are characterized
by the expression of chimeric fusion oncogenes, most commonly
EWS-FLI1
(Kovar, 2005). EWS-FLI1 transforms NIH-3T3 fibroblasts
and its knockdown in
ESFT cells dramatically inhibits tumorigenicity
(Hu-Lieskovan et al., 2005). In
contrast, EWS-FLI1 induces a p53-dependent cell cycle
arrest in primary human
fibroblasts and loss of p16
INK4A
is required
for the transformation of primary
murine fibroblasts (Deneen and Denny, 2001; Lessnick et al., 2002). Appropriate
suppression of innate tumor suppressor
pathways is, therefore, necessary for
EWS-FLI1–mediated
malignant transformation. Unfortunately, evaluation of
primary
ESFT samples has, thus far, yielded little insight into the
mechanism of
this inactivation in vivo. Only 25% of ESFT cases
exhibit either mutation of p53 or
deletion of the p16
INK4a
/ARF
locus, and although these patients exhibit a worse
prognosis,
they clearly represent a minority of cases(Huang et al., 2005).
Polycomb group family proteins are central players in the maintenance
of
stem cell self-renewal and pluripotency as well as in the
control of cellular
differentiation and development (Bracken et al., 2006; Lee et al., 2006; Tolhuis et
al., 2006).
Polycomb proteins assemble into large complexes, termed PRC1
and
PRC2, to repress transcription through modulation of the
chromatin structure and
their expression is frequently deregulated
in cancer (Gil et al., 2005; Molofsky et
6
al., 2004; Valk-Lingbeek et al., 2004). The PRC1 gene bmi-1 was first identified
as an oncogene that collaborates with c-myc in a murine model
of
lymphomagenesis (Haupt et al., 1991; Jacobs et al., 1999b). Studies of bmi-1
knockout mice later
showed that BMI-1 regulates the self-renewal of
hematopoietic,
neural, and neural crest stem cells (Gil et al., 2005).
Mechanistically,
BMI-1 maintains stemness and prevents premature
cellular senescence
in large part through transcriptional repression of Cdkn2a
(Jacobs et al., 1999a).
The CDKN2A locus encodes p16
INK4a
and p14
ARF
, genes
that contribute
to cell cycle regulation and apoptosis through modulation of
the
retinoblastoma and p53 pathways. In somatic stem cells,
BMI-1 functionally
inhibits both pathways thereby supporting
self-renewal and immortality. Down-
regulation of BMI-1 expression
during cellular differentiation is associated with
the release
of this inhibition.
In addition to c-myc, bmi-1 cooperates with the leukemia-associated
translocations E2a-Pbx1 (Smith et al., 2003) and Hoxa9-Meis1 (Lessard and
Sauvageau, 2003)in murine
leukemogenesis, and it has also been implicated in
the origin
of nasopharyngeal carcinoma (Song et al., 2006), neuroblastoma (Cui
et al., 2007; Nowak et al., 2006), and
medulloblastoma (Leung et al., 2004;
Wiederschain et al., 2007). Moreover, the EWS-FLI1–related
protein TLS-ERG
can immortalize primary human hematopoietic
stem cells that have up-regulated
endogenous BMI-1 expression (Warner et al., 2005). Finally, the self-renewal
and tumorigenicity of leukemia,
neuroblastoma, and breast cancer stem cells has
7
been linked
to BMI-1 function (Cui et al., 2006; Lessard and Sauvageau, 2003;
Liu et al., 2006b). Thus, BMI-1 is expressed by
and functions as an oncogene in
many types of human cancer.
Importantly, although direct transcriptional
repression of the
CDKN2A locus contributes to the oncogenic activity of BMI-1
in
cellular models (Jacobs et al., 1999b), recent studies have suggested that
other
mechanisms of BMI-1–mediated tumor promotion exist
and that these are
functionally independent of CDKN2A repression
(Bruggeman et al., 2007; Datta
et al., 2007; Guo et al., 2007b; Wiederschain et al., 2007).
In this study, we have investigated whether BMI-1 functions
as an
oncogene in ESFT. Our findings confirm that BMI-1 is highly
expressed by ESFT
cells and that it promotes anchorage-independent
growth in vitro and tumor
formation in vivo. Furthermore, our
data also show that the mechanism of BMI-1–
mediated tumorigenicity
in ESFT is, at least in part, independent of CDKN2A
repression
and that BMI-1 regulates pathways involved in cell differentiation
and
development as well as cell adhesion. In particular, these
data support the
hypothesis that BMI-1 expression is central
to the pathogenesis of ESFT and that
modulation of cell adhesion
pathways contributes to BMI-1–mediated
tumorigenicity
in this tumor family.
8
Chapter 2 Results
BMI-1 is highly expressed in Ewing’s Sarcoma cell lines and primary tumors.
Expression microarray data generated from primary tumors and ESFT cell
lines were analyzed for BMI-1 expression. As shown in figure 2A, BMI-1
expression was variable but detectable in all samples. These data were
corroborated by QRT-PCR analysis of an independent cohort of primary tumors
and cell lines (Figure 2B). Non-transformed control cells, consisting of primary
human fibroblasts (MRC5), bone marrow–derived mesenchymal stromal cells
(MSC), and human embryonic stem cells (hESC), were similarly evaluated. To
ensure equivalent extracellular environmental stimuli, all cells were collected
during logarithmic growth phase and, with the exception of the hESC, were
grown in the same medium (RPMI with 10% fetal bovine serum) for 24 hours
prior to harvesting. Western blot (Figure 2C) and immunohistochemical analysis
(Figure 2D) confirmed BMI-1 protein expression in ESFT cell lines and primary
tumors, respectively. Histological evaluation of 67 ESFT tumor biopsies revealed
BMI-1 to be diffusely and robustly expressed by tumor cells in 49 cases (73%),
whereas endothelial cells and infiltrating lymphocytes were negative for the
protein (Figure 2D, case I and II). In 18 cases (27%), only rare and/or very
weakly stained BMI-1–positive tumor cells were detected (Figure 2D, case III).
In summary, using both RNA and protein studies, we have found that BMI-
1 is highly expressed by the vast majority of ESFT cells. We are currently
9
investigating the potential clinical and/or biological significance of high versus
low-level BMI-1 expression in primary ESFT.
10
Figure 2. Ewing tumors (ESFT) express BMI-1. A, gene expression profiling using Affymetrix
U133A GeneChips detects expression of BMI-1 (Pcgf-4; Probeset ID 202265_at) in 94 primary
ESFT and 10 ESFT cell lines. Dashed lines, median expression levels; bars, interquantile ranges.
B, QRT-PCR of 20 primary ESFT and 6 ESFT cell lines confirms detectable but variable
expression of BMI-1 in ESFT. Expression in nonmalignant H9 human embryonic stem cells
(hESC), four primary human bone marrow–derived mesenchymal stromal cell cultures (MSC1–
MSC4), and MRC5 lung embryo fibroblasts is uniformly low. Expression levels were repeated in
triplicate and normalized relative to the median expression of GAPDH and ACTIN in the same
sample. C, Western blot analysis detects BMI-1 protein in ESFT cell lines whereas expression in
normal fibroblasts (MRC5) and MSC is very low to undetectable. The variability of protein
expression in both ESFT and nonmalignant cell lines correlates with transcript expression in B. D,
immunohistochemical analysis of 67 ESFT biopsy samples reveals that in 49 cases, tumor cells
are highly positive for BMI-1 (e.g., case I and case II). Unlike tumor cells, endothelial cells in case
I (arrow) and infiltrating lymphocytes in case II (circled) are negative. In 18 cases (e.g., case III),
BMI-1 is weakly expressed or is only expressed by rare cells.
11
Knockdown of BMI-1 does not affect ESFT cell proliferation or death
BMI-1 knockdown does not induce ESFT cell death or CDKN2A
expression. It has been previously shown that BMI-1 promotes the proliferation of
normal human fibroblasts (Guo et al., 2007a), and that loss of BMI-1 in multiple
human cancer cell lines induces cell death (Liu et al., 2006a). To determine if
BMI-1 promotes the survival and/or proliferation of ESFT cells, we assessed the
effects of altered BMI-1 expression on ESFT cells grown in standard culture
conditions. Two different siRNA sequences were used and both sequences
effectively repressed BMI-1 in ESFT and in MCF-7 breast cancer cells (Figure
3A). In contrast to MCF-7 cells, in which BMI-1 knockdown was found to
significantly inhibit both proliferation and survival, BMI-1 knockdown had no effect
on the growth of ESFT cells (Figure 3B). In corroboration with these findings, we
also found that whereas over-expression of BMI-1 promoted the proliferation of
MRC5 fibroblasts, it had no effect on ESFT cell proliferation (data not shown).
12
Figure 3. BMI-1 knockdown does not affect ESFT cell proliferation or death. A, QRT-PCR
confirms the knockdown of BMI-1 following transfection of siRNA oligonucleotides (siBMI1A and
siBMI1B) compared with mock or control (siNS) transfected cells. ESFT cell lines—A4573,
StaET8.2, TC71, and MCF7: positive controls, breast cancer cell line. RNA was harvested from
cells 48 h posttransfection and gene expression normalized to GAPDH expression levels in the
same sample. Columns, averages of three replicate experiments; bars, SD. B, siRNA-mediated
knockdown of BMI-1 significantly inhibits growth of MCF7 but not ESFT cells. Cells transfected
with siRNA oligonucleotides were counted daily for 4 days and the number of viable cells plotted.
BMI-1 knockdown (siBMI1) counts were compared at each time point to control (siNS) transfected
cells. Points, average counts of three replicate experiments; bars, SD. Data is shown for siBMI1B.
Equivalent results were obtained for the siBMI1A sequence (data not shown). C, QRT-PCR
analysis shows no effect of BMI-1 knockdown on CDKN2A expression in serum-free culture.
Stably transduced A4573 cells (shBMI1 and shNS control) were grown in serum-free conditions
for 48 h prior to RNA isolation. Columns, average of replicate experiments with gene expression
expressed relative to GAPDH in the same samples; bars, SD. D, knockdown of BMI-1 has no
effect on ESFT cell proliferation or death in cells grown in serum-free conditions. A4573 cells
stably transduced with a BMI-1 hairpin construct (shBMI1) and grown in serum-free conditions for
48 h show no change in cell death (left; fluorescence-activated cell sorting analysis of Annexin-
V/propidium iodide–stained cells) or cell proliferation (right; fluorescence-activated cell sorting
analysis of fixed, propidium iodide–stained cells).
13
Because BMI-1 has been reported to exert its effects through the
repression of CDKN2A and its protein products p16
INK4A
and p14
ARF
, we
reasoned that the lack of effect of BMI-1 modulation on ESFT cell proliferation
and death may be a consequence of CDKN2A deletion in the cell lines. Using a
combination of QRT-PCR and western blot, we found that A4573, StaET8.2, and
StaET7.2 express the CDKN2A transcript and both A4573 and StaET8.2 express
the p16 protein (Figure 4A and 4B). Genomic real-time PCR (Labuhn et al.,
2001) revealed that TC71, TC32, and TC252 cells all have homozygous loss of
CDKN2A, StatET8.2, and StatET7.2 are wild-type for the locus, and A4573 is
hemizygously deleted (Figure 4C and 4D) Thus, BMI-1 knockdown does not
inhibit ESFT cell growth irrespective of their p16 status. Next, we set out to
determine whether BMI-1 represses CDKN2A transcript expression in p16
INK4A
-
wildtype ESFT cells. Unexpectedly, we found that although BMI-1 repressed the
expression of CDKN2A/p16
INK4A
in human MSC and Tera2 embryonic carcinoma
cells, no significant or consistent effect was observed in ESFT cell lines (Figure 5
A, 5B, and 5C). The alternate protein product encoded by the CDKN2A locus,
p14
ARF
, was not expressed to detectable levels by ESFT (data not shown). BMI-
1–dependent repression of a p21-retinoblastoma pathway has recently been
implicated in the control of embryonic neural stem cell self-renewal (Fasano et
al., 2007). To determine if BMI-1 may be targeting p21 rather than p16 in ESFT
cells, we also evaluated CDKN1A expression and p21 protein levels following
BMI-1 gain and loss of function. p21 protein levels were unaffected by BMI-1
14
knockdown in all six ESFT cell lines tested whereas CDKN1A was up-regulated
in A4573 cells and down-regulated in StaET8.2 cells (data not shown). No
change in pRB-phosphorylation was observed in any ESFT cell line following
BMI-1 knockdown (Figure 5D). Together, these data indicate that BMI-1 does not
significantly modulate either CDKN2A or CDKN1A expression in ESFT and the
failure of BMI-1 to promote ESFT proliferation and/or survival is not a
consequence of CDKN2A/p16 loss.
15
Figure 4. CDKN2A/p16 status of ESFT cell lines A. Quantitative RT-PCR analysis of non-
malignant (hESC, huMSC, MRC5, 293FT) and ESFT cell lines reveals that CDKN2a is not
expressed by TC71, TC32, and TC252 ESFT cells. B. Western blot demonstrates that among six
ESFT cell lines tested, only A4573 and StaET8.2 express detectable levels of the p16 protein. C.
Genomic PCR amplification of exon 1a of the CDKN2a locus relative to GAPDH amplification
confirms that this p16-encoding exon is homozygously deleted in TC71, hemizygously deleted in
A4573 and wild-type in StaET8.2 cell. D. Summary of the Exon 1a status of ESFT cell lines as
evaluated by genomic real-time PCR.
16
Figure 5. BMI-1 modulation does not affect CDKN2A levels or p16-RB pathway activation. A.
Quantitative RT-PCR analysis of cell lines transfected with BMI1-targeted siRNA oligonucleotides
confirms knockdown of BMI-1 but no significant change in CDKN2A expression in ESFT cells. In
contrast, and as predicted, CDKN2A expression was induced following BMI-1 knockdown in
human embryonal carcinoma cells (TERA2). Expression values were calculated relative to
GAPDH in the same sample and then normalized to equivalently treated control (siNS) cells.
Histograms show the mean % expression and error bars represent the standard deviations for
triplicate experiments. B. siRNA-transfected ESFT cells (siBMI1A (A), siBMI1B (B)) show reduced
expression of BMI-1 by western blot compared to control cells (siNS (N)) but no concomitant
increase in p16 is observed. No significant change in expression of the BMI-1 related polycomb
protein MEL-18 is observed following BMI-1 knockdown in either ESFT or MCF7 cells. C. Forced
over-expression of BMI-1 (pBp-BMI1) in ESFT cells does not lead to down-regulation of p16. In
contrast, over-expression of BMI-1 in human MSC leads to the predicted reduction in p16
expression. D. Western blot of total cell lysates obtained 48 hrs post-siRNA transfection reveals
that knockdown of BMI-1 in ESFT cell lines has no impact on pRB phosphorylation. E.
Retinoblastoma protein (RB) is largely hyper-phosphorylated in ESFT cells. MRC5 fibroblasts and
ESFT cells were grown in serum free (serum -) or 10% FBS containing (serum +) media and
subconfluent cell lysates subjected to western blot analysis. As shown, RB is largely
phosphorylated (inactive) (P-RB) in ESFT cells in both the presence and absence of serum.
Although the levels of phosphorylated RB are reduced in serum free culture, there is also an
overall reduction in total RB.
17
Figure 5 continued
18
The mechanism of BMI-1–mediated repression of p16
INK4A
has been
shown to involve direct binding of the CDKN2A promoter and to depend on the
presence of a functional retinoblastoma protein (pRB; (Kotake et al., 2007)).
ESFT cells display hyper-phosphorylated (inactive) pRB when grown in standard
culture conditions and although serum withdrawal leads to a reduction in pRB
phosphorylation, this is accompanied by a reduction in total pRB expression
(Figure 5E). To determine if BMI-1 knockdown is able to effect changes in ESFT
growth in conditions in which pRB may be relatively more active, we repeated
cell growth assays in serum-free conditions. Consistent with our earlier findings,
we observed no significant effects of BMI-1 knockdown on CDKN2A expression
level, cell proliferation, or cell death (Figures 3C and 3D; data not shown).
BMI-1 promotes anchorage independent growth
BMI-1 promotes the anchorage-independent growth and tumorigenicity of
Ewing tumor cells. Although BMI-1 loss did not alter ESFT proliferation or death,
we observed that knockdown dramatically altered the morphologic characteristics
of A4573 cells in vitro. Whereas these cells normally grow as three-dimensional
clusters, knockdown of BMI-1 reproducibly resulted in conversion to growth as
adherent cellular monolayers (Figure 6A). We therefore reasoned that BMI-1
might contribute to anchorage-independent colony formation. To assess whether
the anchorage-independent growth of ESFT cells in vitro is affected by altered
BMI-1 expression levels, we performed soft agar assays of cells that were
genetically modified to express altered levels of BMI-1. As shown, knockdown of
19
BMI-1 inhibited colony formation (Figure 6B), whereas over-expression of BMI-1
led to a more rapid formation of macroscopic colonies and an increase in
macroscopic colony number (Figure 6C). Confirmation of the specificity of the
effects of BMI-1 knockdown was achieved by demonstrating equivalent effects
using a second shRNA sequence (data not shown).
20
Figure 6. BMI-1 promotes the anchorage-independent growth of ESFT cells. A, phase contrast
images of A4573 cells growing in standard culture conditions. siRNA-mediated knockdown of
BMI-1 (siBMI1) causes A4573 cells to flatten whereas Lipofectamine-treated and control
transfected (siNS) cells continue to grow as three-dimensional clusters. B, stable knockdown of
BMI-1 (shBMI1B) impairs colony formation of ESFT cells in soft agar. Images are of macroscopic
colonies on the day at which control cell (shNS) media was depleted for each of the three
respective cell lines. Each well is representative of six replicate wells for each condition. Similar
results were obtained with a second BMI-1–targeted shRNA construct (data not shown). C, ESFT
cells transduced to overexpress BMI-1 (pBp-BMI1) have a reduced time to macroscopic colony
formation and increased colony number in soft agar when compared with control, empty vector–
transduced cells (pBp-V). Images are of macroscopic colonies on the day at which pBp-BMI1
media was depleted for each of the three respective cell lines. Each well is representative of six
replicate wells for each condition.
BMI-1 promotes tumorigenicity in vivo
To determine if BMI-1 promotion of ESFT colony formation in vitro
correlates with in vivo tumorigenicity, we evaluated xenograft tumor formation in
NOD-SCID mice. As shown in Figures 7A and B, the rate of engraftment of TC71
tumor cells directly correlated with BMI-1 expression levels. For shBMI1 cells, the
median time to measurable tumor was 16 days compared with only 13 days for
shNS cells (P < 0.05). In contrast, BMI-1–overexpressing cells formed tumors
within 10.5 days compared with 15 days for empty vector–transduced cells (P=
21
0.005). Similarly, whereas A4573 shNS control cells formed tumors with a
median time of 15 days, only one of five shBMI1-recipient mice developed a
tumor and this did not appear until 37 days postimplantation (data not shown). To
confirm that the xenografts did not form from cells that had escaped genetic
modification, variable expression of BMI-1 was confirmed in the excised tumors
(Figure 7C).
22
Figure 7. BMI-1 promotes ESFT tumorigenicity in vivo. A, NOD-SCID mice injected with TC71
cells that express reduced levels of BMI-1 (shBMI1) show delayed engraftment and slowed in
vivo growth compared with mice injected with control (shNS) transduced cells (two-tailed paired t
test, P < 0.001). B, time to tumor cell engraftment is decreased and tumor growth enhanced in
NOD-SCID mice injected with TC71 cells that over-express BMI-1 (pBp-Bmi1) compared with
empty vector (pBp-V) transduced cells (two-tailed paired t test, P = 0.01). C, QRT-PCR analysis
of RNA harvested from tumor xenografts confirms differential expression of BMI-1.
23
BMI-1 knockdown effects significant changes in developmental and cell adhesion
pathways.
Having established that BMI-1 promotes ESFT tumorigenicity in the
absence of CDKN2A modulation, we initiated studies to identify novel effectors of
BMI-1 action. To achieve this, we performed expression profiling of A4573 cells
following acute knockdown of BMI-1 and found that nearly 900 known genes
were significantly affected (P < 0.01), and 245 of these were altered at least 2-
fold (Figure 8; GEO accession series no. GSE12064). Assessment of the gene
ontology designations of these BMI-1–responsive genes found several pathways
to be significantly overrepresented, in particular, pathways involved in cellular
development as well as adhesion and invasion (Table 1). In addition, whereas
many genes in these overrepresented pathways were induced by BMI-1
knockdown (e.g., NOTCH1, WNT5A, TIMP2, and TIMP3), others were down-
regulated (e.g., COL5A2, COL1A2, and ICAM2) demonstrating that although
BMI-1 is known to function as a transcriptional repressor, inhibition of its
expression does not exclusively lead to gene induction and genes that are
indirectly regulated by BMI-1, either up-regulated or down-regulated, may
contribute to its function as an oncogene.
24
Figure 8. Gene expression profiling of A4573 BMI-1 knockdown (siBMI1) and control (siNS) cells.
Heat map representation of the expression patterns for 900 genes affected as a result of acute
BMI-1 knockdown in A4573 ESFT cells (p<0.01).
25
Table 1. Gene ontology biological processes significantly affected by BMI-1 knockdown in A4573
ESFT cells.
In an attempt to identify the downstream genes that are most likely to
mediate tumorigenicity, we compared our data to a previously published analysis
of BMI-1 knockdown in DAOY medulloblastoma cells (Wiederschain et al., 2007).
Raw data from this study was extracted from the National Center for
Biotechnology Information GEO database (series no. GSE7578) and processed
using the same methodology used for analysis of our ESFT data (described in
Materials and Methods). Comparison of the two independent gene lists reveals
that expression of 101 genes was significantly and commonly altered by BMI-1
knockdown in both A4573 and DAOY cells (Figure 9). Importantly, whereas
3/5/10 11:32 AM Cancer Research -- Douglas et al. 68 (16): 6507 Table BL1
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Table 1. Gene ontology biological processes significantly affected by BMI-1 knockdown in A4573 ESFT
cells
No. Gene ontology biological process Douglas et al.
(P)
Overlap with Wiederschain et
al.
1 Development: neurogenesis in general 3.2E–07
*
2 Proteolysis: connective tissue degradation 9.9E–05
*
3 Cell adhesion: cell-matrix interactions 1.0E–04
*
4 Reproduction: progesterone signaling 2.5E–04 NS
5 Cell adhesion: amyloid proteins 2.6E–04
*
6 Signal transduction: WNT signaling 4.2E–04 NS
7 Cytoskeleton: regulation of cytoskeleton
rearrangement
5.5E–04 NS
8 Proteolysis: ECM remodeling 2.3E–03
*
9 Development: skeletal muscle development 2.3E–03
*
10 Cell adhesion: integrin-mediated cell-matrix
adhesion
2.6E–03 NS
11 Signal transduction: neuropeptides signaling
pathways
3.1E–03 NS
12 Signal transduction: NOTCH signaling 4.5E–03
*
Abbreviation: NS, nonsignificant overlap.
*
Processes in which statistically significant overlap (P < 0.01) exists in BMI-1–modulated genes between
ESFT and medulloblastoma cells (19).
26
A4573 cells express p16, DAOY are p16-null, further supporting the designation
of these BMI-1 targets as p16-independent (Fruhwald et al., 2001). Moreover,
DAOY cells were studied following shRNA transduction and antibiotic selection,
whereas A4573 cells were analyzed following acute siRNA-mediated knockdown.
Thus, the common effects on gene expression cannot be attributed to the
consequences of experimental design. Rather, these 101 genes are likely to
represent true BMI-1 targets. A high degree of overlap in overrepresented
biological processes between the two model systems is also observed, with the
same developmental and cell adhesion genes featuring prominently (Table 1). To
ensure that the overlap between the gene sets was not the result of chance, we
computed the probability of obtaining the observed number of overlaps under a
hypergeometric distribution and confirmed the degree of overlap to be highly
statistically significant (P < 0.001).
27
Figure 9. Comparison of gene expression pattern between BMI-1 knockdown cells in ESFT and
medulloblastoma identified 101 overlapping BMI-1 targets. B. Left. Venn diagram illustration of
101 genes affected as a result of BMI-1 knockdown in A4573 ESFT (Douglas et al 2008) cells
and DAOY medulloblastoma cells (Wiederschain et al 2007) (p<0.001). 56 of the genes were
commonly up-regulated as a result of BMI-1 knockdown in these two independent cell lines. 45 of
the genes were down-regulated in the two cell lines. Right. Gene expression pattern of 101 over-
lapping putative BMI-1 target genes is depicted in a heat map representation.
In view of the striking overrepresentation of genes involved in cellular
adhesion, we evaluated the consequences of BMI-1 modulation on the adhesion
of A4573 cells in vitro. As shown in figure 10A, adhesion is promoted by BMI-1
over-expression and inhibited by BMI-1 knockdown in these cells. To validate
that adhesion genes identified by microarray analysis are bona fide targets of
BMI-1, we performed QRT-PCR and western blot analysis to assess the
28
expression levels of NID1 and VEZT, and their respective protein products,
nidogen 1 and vezatin. Although expression of both genes was consistently and
reproducibly altered by BMI-1 knockdown in ESFT cell lines (Figure 10B), only
nidogen 1 expression was altered at the level of protein expression (Figure 10C).
Nidogen 1 is a cell adhesion protein and integral component of basement
membranes (Kohling et al., 2006). To determine if nidogen 1 levels affect ESFT
cell adhesion, we examined the consequences of NID1 knockdown. As shown in
figure 10D, NID1 knockdown accelerated ESFT cell adhesion, suggesting that
repression of nidogen 1 may be functionally important in BMI-1–mediated
adhesion and tumorigenicity. More extensive studies are now required to test this
possibility.
29
Figure 10. BMI-1 accelerates ESFT cell adhesion in vitro. A, A4573 cells were plated in 96-well
plates and monitored for adhesion. Cells with stable knockdown of BMI-1 (shBMI1) show reduced
adhesion relative to control cells (shNS; top), whereas adhesion is accelerated in BMI-1–
overexpressing cells (pBp-BMI1) in comparison with empty vector cells (pBp-V; bottom). Points,
mean absorbance of crystal violet taken up by adherent cells in at least seven replicate wells from
duplicate experiments at 30-min intervals. B, QRT-PCR validates that two cell adhesion genes
identified by microarray analysis are truly BMI-1–responsive. Note that although basal levels of
the two genes differ, knockdown of BMI-1 leads to significant up-regulation of NID1 and down-
regulation of VEZT in all three ESFT cell lines. C, Western blot confirms the up-regulation of
nidogen 1 protein in A4573 cells following BMI-1 knockdown (siBMI1A and siBMI1B) and down-
regulation in the presence of BMI-1 overexpression (pBp-BMI1). D, A4573 cells were transfected
with siNID1 or siNS control oligonucleotides and knockdown confirmed after 48 h by QRT-PCR
(left). Transfected cells were plated in 96-well plates and adhesion measured as described in A.
Reduced expression of NID1 accelerates adhesion.
30
Figure 10 continued
31
WNT5A mediates ESFT cell adhesion downstream of BMI-1
Members of the WNT gene family have been implicated in many human
cancers (Polakis, 2000). Interestingly, gene expression profiling on ESFT BMI-1
knockdown cells enabled us to discover WNT5A, a member of the non-canonical
WNT gene family, to be among the list of genes up-regulated in A4573 BMI-1
knockdown ESFT cells (Figure 9). To verify our expression microarray data, we
found that knockdown of BMI-1 indeed resulted in up-regulation of WNT5A at the
transcript level in both TC71 and A4573 ESFT cells (Figure 11A). We will further
conduct experiments to confirm WNT5A protein expression in both cells lines.
Although activation of WNT signaling pathway is generally thought be to
oncogenic, emerging data suggests WNT5A can have tumor suppressing roles
(McDonald and Silver, 2009). The precise mechanism underlying WNT5A-
mediated tumor suppression is enigmatic. Studies have shown that WNT5A can
inhibit cancer progression by promoting cell adhesion (Medrek et al., 2009). This
leads us to hypothesize that WNT5A may play a critical role in modulating cell
adhesion downstream of BMI-1. In support of our hypothesis, we found that
modulation of WNT5A protein level in culture media using conditioned WNT5A
medium resulted in marked change in the adhesive property of A4573 ESFT cells
much like BMI-1 knockdown cells (Figure 11B). Whether or not alteration of
adhesion instigated by WNT5A has an impact on the tumorigenicity of ESFT cells
remains a pressing question. Future studies will be conducted to verify the role
32
of WNT5A as a tumor suppressor in ESFT, as well as its function in promoting
cell adhesion downstream of BMI-1.
33
Figure 11. BMI-1 alters the adhesion property of ESFT through WNT5A repression. A. QRT-PCR
analysis of WNT5A expression in A4573 and TC71 ESFT cells following BMI-1 knockdown by
RNAi. WNT5A transcript is up-regulated as a result of BMI-1 knockdown. B. Upper panel depicts
phase contrast images of A4573 cells transfected with lipofectamine, non-silencing siRNA(siNS),
BMI-1 siRNA. Bottom panel shows phase contrast images of A4573 cells treated with control
conditioned cell media and WNT5A conditioned media. Treatment of A4573 cells with WNT5A
conditioned media or siRNA against BMI-1 (siBMIB) leads to a similar change in cell morphology.
34
Mechanism of BMI-1-mediated silencing of NID1 and WNT5A
Members of the polycomb group family complexes PRC1 and PRC2
coordinately silence gene transcription through chromatin modification and DNA
methylation via recruitment of DNA methyl-transferase (DNMT) (Hernandez-
Munoz et al., 2005; Negishi et al., 2007). To determine if WNT5A and NID1
promoters are repressed as a result of BMI-1 mediated hyper-methylation, we
exposed ESFT cells TC71 and A4573 to 5-AZA-dC, a demethylating reagent
which inhibits the activity of DNMT, and monitored the expression level of both
genes. We found that after 72h treatment with 5-AZA-dC, WNT5A and NID1
transcripts were significantly increased (Figure 12A). This suggests
hypermethylation of gene promoters partly contribute to NID1 and WNT5A gene
silencing.
Recent studies on epigenetic stem cell signature in cancer have
suggested that polycomb group protein targets are highly susceptible to promoter
DNA hypermethylation comparing to non-polycomb group protein targets
(Widschwendter et al., 2007). In order to identify putative BMI-1 targets that are
preferentially hypermethylated in ESFT, we collaborated with Dr. Peter Laird of
the USC Epigenome Center to examine the methylation status of over 1,500
polycomb group protein target genes in ESFT cells using Illumina GoldenGate
technology (Bibikova et al., 2006; Lee et al., 2006). Intriguingly, among the list of
genes evaluated in BMI-1 over-expressing and BMI-1 knockdown ESFT cells, we
discovered that over-expression of BMI-1 in A4573 ESFT cells promoted
35
methylation of the WNT5A gene, while knockdown of BMI-1 resulted in a
significant decrease in WNT5A DNA methylation (Figure 12B). Our preliminary
analysis on the methylation status of polycomb group protein targets implies that
high level of BMI-1 expression in ESFT can lead to hypermethylation and
inactivation of WNT5A. To verify WNT5A promoter is indeed silenced as a result
of hypermethylation, we will perform bisulfite sequencing of the WNT5A promoter
region to validate the result generated by the Illumina GoldenGate assay. It is
noteworthy that the list of polycomb group protein targets evaluated here was
originally identified based on their association with various members of the PRC2
complex in hESC cells (Lee et al., 2006; Widschwendter et al., 2007). Therefore,
to ensure all the polycomb group protein targets are accounted for, a more
extensive study will be required to fully evaluate the methylation status of all
polycomb group protein target genes including those that are bound by the PRC1
complex.
36
Figure 12. Hyper-methylation of NID1 and WNT5A promoters in ESFT correlates with BMI-1
expression. A. QRT-PCR analysis shows dose-dependent de-repression of NID1 and WNT5A
gene expressions in TC71 cells treated with low (+;0.625 µg/mL) and high (++;2.5µg/mL) dose of
5AZA-dC for 72 hours. B. BMI-1 promotes methylation of WNT5A. WNT5A DNA methylation
analysis using Illumina GoldenGate platform on BMI-1 over-expressing (red) and BMI-1
knockdown (blue) A4573 ESFT cells.
37
Chapter 2 Discussion
We have found that BMI-1 is highly expressed by ESFT cells and that it
promotes anchorage-independent growth and in vivo tumorigenicity. Importantly,
our studies reveal that these tumorigenic properties of BMI-1 are modulated
independent of CDKN2A repression, indicating that novel mechanisms of BMI-1
oncogenic activity exist. In fact, in contrast to normal mesenchymal stem cells,
altering expression levels of BMI-1 has no significant or consistent effect on
CDKN2A or p16
INK4A
expression in ESFT. Although this may be an artifact of in
vitro culture, it is also possible that nonfunctional retinoblastoma family proteins
prevent BMI-1–mediated repression of CDKN2A in these cells (Kotake et al.,
2007). Although previous reports have shown that pRB is only rarely mutated in
ESFT (Kovar et al., 1997; Maitra et al., 2001), recent work suggests that pRB
function may be inactivated by EWS-FLI1 itself (Hu et al., 2008). Further studies
are now required to determine if pRB inactivation contributes to the dissociation
of BMI-1 from p16
INK4A
regulation in ESFT. In addition to Rb, polycomb-mediated
silencing of CDKN2A locus depends on the interplay among several molecular
factors. It has been shown that recruitment of BMI-1 to CDKN2A requires the
presence of PRC2 complex and that loss of EZH2, a catalytic component of the
PRC2 complex, leads to derepression of the CDKN2A locus (Agger et al., 2009;
Barradas et al., 2009; Bracken et al., 2007). There is also evidence to suggest
that JMJD3 (Agger et al., 2009; Barradas et al., 2009), a histone demethylase,
and MLL1 (Dhawan et al., 2009), a member of the trithorax group of proteins, are
38
implicated in the regulation of CDKN2A expression. Further, homologues of
various components of polycomb complex may substitute and compensate the
function of one another. For example, EZH1, a homologue of EZH2, may
mediate methylation on histone H3 lysine 27 in the same manner as EZH2 (Shen
et al., 2008). Indeed, our study shows that knockdown of BMI-1 has no effect on
the expression of MEL18 (a homologue of BMI-1) and EZH2 (Figure 5B, 5C, and
data not shown), implicating that loss of BMI-1 alone may not be sufficient to
induce CDKN2A derepression while other components of PRC1 and PRC2
remain intact and functional.
Although the histogenesis of ESFT remains a mystery, recent studies
implicate somatic stem cells as cells of origin (Meltzer, 2007). Given that most
somatic stem cells express high-levels of BMI-1 and that expression diminishes
during differentiation (Hosen et al., 2007), it is possible that the high level of BMI-
1 expression we observe in ESFT cells is an inherent feature of their cellular
origin. Alternatively, expression of the EWS-FLI1 fusion oncogene may be able,
in some cell types, to induce BMI-1 as was recently shown in NIH-3T3 cells
(Zwerner et al., 2008). Cell type– and differentiation state–appropriate
experimental models are now required to test which of these situations exists in
the initiation of ESFT. EWS-FLI1–mediated transformation of primary fibroblasts
requires the inactivation of p16-retinoblastoma and/or p53 pathways (Deneen
and Denny, 2001; Lessnick et al., 2002). We speculate that BMI-1–mediated
repression of CDKN2A may confer cellular tolerance to EWS-FLI1 in the ESFT
39
cell of origin and that this epigenetic inactivation of tumor suppressor pathways
could explain the relatively low incidence of secondary genetic mutations in
primary ESFT (Huang et al., 2005). In support of this, we find that BMI-1 levels
are, in general, higher among primary tumors compared with ESFT cell lines
(Figure 2A), suggesting that mutations in p16
INK4a
and/or p53 that are more
commonly present in cell lines (Kovar et al., 1993; Kovar et al., 1997) may at
least partially compensate for BMI-1 expression. It has been previously
documented that p16
INK4A
loss in lung tumors correlates with low BMI-1
expression (Vonlanthen et al., 2001). We are now testing whether there is a
relationship between BMI-1 expression and p16
INK4A
and/or p53 status in primary
ESFT and whether differences exist in clinical presentation or outcome between
tumors that express high versus low levels of BMI-1.
We have shown that altering BMI-1 expression affects the ability of both
p16-null and p16-positive cells to form anchorage-independent colonies in vitro
and tumors in vivo. In corroboration with our findings, several recently published
reports have revealed that, in cooperation with other oncogenic lesions such as
mutated epidermal growth factor receptor or H-RAS, BMI-1 can transform both
CDKN2A wild-type and CDKN2A null cells (Bruggeman et al., 2007; Datta et al.,
2007). In addition, BMI-1 knockdown in p16-null DAOY medulloblastoma cells
significantly impedes tumor formation in vivo (Fruhwald et al., 2001;
Wiederschain et al., 2007). Thus, although initial studies of BMI-1 implicated the
repression of the p16INK4A/p14ARF-encoding CDKN2A locus as the primary
40
mechanism of oncogenic action (Jacobs et al., 1999a; Jacobs et al., 1999b),
more recent data from our lab and others show a pivotal role for p16-independent
mechanisms.
In order to identify potentially novel downstream targets of BMI-1, we
performed gene expression profiling of ESFT cells following BMI-1 knockdown
and compared BMI-1–responsive genes to those genes similarly affected by
BMI-1 knockdown in human medulloblastoma cells (Wiederschain et al., 2007).
Although a significant subset of genes was commonly regulated by BMI-1 in both
tumor types, others were uniquely altered in only one of the model systems. This
implies that although some biological pathways are shared among different tumor
types, it is likely that at least some downstream effectors of BMI-1 differ among
tumors of different cellular origins. Nevertheless, our findings show that
significant commonalities exist. In particular, direct comparison between ESFT
and medulloblastoma cells reveals that alterations in cell adhesion and
extracellular remodeling processes are highly overrepresented and common to
both tumor types. For the current study, we have validated that expression of the
basement membrane protein nidogen 1 and the non-canonical WNT gene
WNT5A are repressed by BMI-1 in ESFT cells. Nidogen 1 acts as a linker
between laminins, collagens, and proteoglycans in the extracellular matrix and
binds to cell surface integrins (Kohling et al., 2006). Interestingly, it has recently
been reported that NID1 is frequently silenced in colon cancer, suggesting that
nidogen 1 may have a role as a tumor suppressor gene, preventing invasion and
41
metastasis (Ulazzi et al., 2007). In the same respect, WNT5A has been shown to
be involved in regulating cell fate, cell adhesion, and motility by initiating diverse
intracellular signaling (Katoh, 2008). Paradoxically, although WNT5A can
function as a tumor promoter, in some cancer it can also function as a tumor
suppressor whose expression is inactivated by aberrant methylation (Da Forno et
al., 2008; Roman-Gomez et al., 2007; Ying et al., 2007; Ying et al., 2008). We
therefore hypothesize that epigenetic inactivation of NID1 and WNT5A and their
biological function are central to BMI-1-mediated ESFT tumorigenicity. In support
of this possibility, we have found that down-regulation of NID1 promotes
adhesion of ESFT cells in vitro, recapitulating the effects of BMI-1
overexpression. Similarly, addition of WNT5A protein to culture media affects the
adhesive property of ESFT cells much alike BMI-1 knockdown cells. Therefore,
we hypothesize that the effect of BMI-1 knockdown on cell adhesion, through
modulation of nidogen 1, WNT5A, and/or other adhesion-related proteins, is
likely to underlie the delay to in vivo tumor engraftment that we observe in ESFT
cells with reduced levels of BMI-1. Consistent with this hypothesis, delayed
engraftment and altered adhesion pathways have also been shown to be a
feature of bmi-1–deficient murine glioma cells (Bruggeman et al., 2007). Thus,
the cumulative evidence suggests that modulation of adhesion molecules such
as nidogen 1 and WNT5A is likely to contribute to the oncogenic function of BMI-
1 in ESFT as well as other tumor types.
42
The molecular mechanism underlying BMI-1 mediated repression of its
putative targets, NID1 and WNT5A, has not been explored. BMI-1 protein, as a
member of the PRC1 complex, mediates gene silencing via chromatin
modification and DNA methylation through interaction with DNA methyl-
trnasferase (Bracken and Helin, 2009; Hernandez-Munoz et al., 2005; Negishi et
al., 2007). We therefore hypothesize that BMI-1 inactivates NID1 and WNT5A
epigentically by promoting their promoter hypermethylation and/or histone 3
lysine 27 trimethylation and/or histone H2A lysine 115 ubiquitylation. In support
of our hypothesis, we observe that modulation of BMI-1 affects the methylation
status of WNT5A. We also have preliminary evidence to suggest that both NID1
and WNT5A promoters are aberrantly methylated in ESFT cells although the
methylation status of the promoters remains to be validated by bisulfite
sequencing. Experiments are ongoing to further elucidate whether or not BMI-1
binds to NID1 and WNT5A promoters to affect their methylation status.
Moreover, We will explore if BMI-1 mediated chromatin modification on H3K27
and/or H2AK119 play a major role in the silencing of NID1 and WNT5A genes.
Finally, polycomb genes, including BMI-1, play a central role in the
repression of differentiation and in the controlled orchestration of normal
development (Lee et al., 2006). Our finding that developmental pathways are
significantly affected by BMI-1 knockdown in ESFT cells suggests that the
embryonic function of BMI-1 is being recapitulated in these undifferentiated tumor
cells. It is particularly noteworthy that both WNT and NOTCH pathway genes are
43
highly affected by BMI-1 knockdown as both of these developmental pathways
have been previously implicated in ESFT growth and tumorigenicity (Baliko et al.,
2007; Uren et al., 2004). Intriguingly, our data also show that among the affected
developmental processes, BMI-1 loss has its most profound effect on genes that
are involved in neural development with BMI-1 knockdown leading to increased
expression of neural markers (Table 1). These findings corroborate recent
documentation of the effects of bmi-1 loss on the phenotype and neural
differentiation capacity of murine gliomas (Bruggeman et al., 2007). One of the
many clinical mysteries surrounding ESFT is the observation that they vary from
highly undifferentiated tumors to tumors with obvious neural features. It is
tempting to speculate that the phenotype of a particular tumor is either (a)
predetermined by the BMI-1 expression level in the parent cell that originally
acquires the EWS-ETS translocation or (b) a consequence of the tumor
microenvironment and its downstream effects on BMI-1 expression. More
extensive studies are required to evaluate these hypotheses.
In summary, we have shown that BMI-1 functions as an oncogene in
ESFT and that it promotes tumorigenicity in a CDKN2A-independent manner,
influencing pathways involved in cell adhesion, differentiation, and development.
Given its central role in the regulation of multiple developmental processes in
normal stem cells, we expect that no single gene will be uniquely responsible for
the oncogenic effects of BMI-1. Nevertheless, our findings support the hypothesis
that regulation of adhesion pathways is central to BMI-1–mediated tumor
44
promotion. Future studies directed at understanding this relationship are likely to
proffer attractive and novel targets for therapeutic intervention that may be
common to the multiple human cancers that deregulate and over-express BMI-1.
45
Chapter 3
BMI-1 cooperates with Yes-Associated Protein, YAP, to suppress cell
contact inhibition in Ewing’s sarcoma
Chapter 3 Introduction
The Ewing’s sarcoma family of tumors (ESFT) is a group of poorly
undifferentiated, highly aggressive bone and soft tissue malignancy inflicting
mostly children and adolescents. Despite multimodal treatment regimens, the
survival rate for patients remains 60-75% for patients with localized disease and
<20% for patients with metastases at diagnosis(Balamuth and Womer, 2010; Ban
et al., 2006). Developing novel therapeutic molecular targets remains challenging
while the molecular nature and the cell of origin of ESFT are beginning to be
unraveled.
The stem cell self-renewal pathways are frequently deregulated in cancer.
In particular, the polycomb group family protein BMI-1 is highly expressed in a
number of malignancies, including ESFT (Douglas et al., 2008). BMI-1 has been
shown to promote stemness and tumorigenicity partly through epigenetic
repression of the CDKN2A locus, inhibiting the expression of cell-cycle inhibitors
p16
INK4A
and ARF, thereby blocking their respective tumor suppressing function
(Jacobs et al., 1999a).
We have previously shown that BMI-1 functions as an oncogene in ESFT,
promoting anchorage independent growth in vitro and tumorigenicity in vivo
independently of CDKN2A repression (Douglas et al., 2008). Consistent with our
46
observation, other groups have revealed that BMI-1 may exert its oncogenic
function by affecting other developmental and growth signaling pathways in
cancer cells irrespective of their CDKN2A status (Bruggeman et al., 2007; Datta
et al., 2007; Wiederschain et al., 2007). More and more data have now emerged
to support other novel functions of BMI-1, including its roles in regulating
oxidative stress, mitochrondrial function, and DNA damage response (Chatoo et
al., 2009; Liu et al., 2009).
Cell contact inhibition is a fundamental tumor suppressing property of
normal cells. Many decades ago, Abercrombie and Heaysman observed that
unlike normal cells, cancer cells have lost the ability to cease proliferation and
movement upon reaching confluence in culture (Abercrombie and Heaysman,
1954). Loss of cell contact inhibition therefore can lead to cancerous outgrowth
and invasion, thereby increasing the malignant potential of cancer cells
(Abercrombie, 1979; Eagle and Levine, 1967; Silletti et al., 1995). In this study,
we have investigated the role of BMI-1 in de-regulating cell contact inhibition.
Using ESFT cells that stably express shRNA oligos against BMI-1, we
discovered that loss of BMI-1 expression restores contact inhibition to ESFT
cells. Although many signaling pathways are involved in mediating the cell
contact inhibition response, inactivation of the Yes-Associated Protein (YAP), a
key downstream effector of the Hippo pathway, has been implicated in both
Drosophila and mammalian cells (Zhao et al., 2008; Zhao et al., 2007).
Deregluation of YAP-mediated signaling has been associated with a number of
47
human malignancies, including hepatocellular carcinoma and medulloblastoma,
and colorectal cancer (Camargo et al., 2007; Fernandez et al., 2009; Liu et al.;
Overholtzer et al., 2006). Intriguingly, our data shows that silencing BMI-1
expression results in loss of YAP protein at high cell density coincident with
induction of contact inhibition. Knockdown of YAP in ESFT cells decrease their
proliferative potential and anchorage-independent growth in soft agar. Together,
these findings suggest that YAP is a novel downstream target of BMI-1 and that
BMI-1-mediated stabilization of YAP renders cancer cells impervious to cell
contact inhibition.
48
Chapter 3 Results
Knockdown of BMI-1 in ESFT restored contact inhibition.
We have previously shown that knockdown of BMI-1 by RNAi in ESFT
(Figure 13A and 13C) has a tremendous impact on ESFT tumorigenicity in vitro
and in vivo (Douglas et al., 2008). To gain further insight on the biological
process underlying BMI-1-mediated tumorigenicity, we examined the role of BMI-
1 in loss of cell contact inhibition in ESFT. We hypothesize that BMI-1 promotes
ESFT growth by deregulating pathways involved in activating cell contact
inhibition. To assess the role of BMI-1 in cell contact inhibition, we monitored the
growth phenotype of BMI-1 knockdown ESFT cells in high-density cell cultures.
As shown in figure 13B, TC71 (p16 null; p53mut) and CHLA9 ESFT (p16wt;
p53wt) cells growing at logarithmic phase were healthy and knockdown of BMI-1
in these cells did not cause any significant phenotypic changes. However, in
post-confluent culture, loss of BMI-1 in TC71 and CHLA9 ESFT cells induced a
dramatic change in cell morphology resembling of dying cells (Figure 13B). To
further characterize the growth inhibiting effect as a result of BMI-1 knockdown in
high-cell density cultures, we measured the effect of BMI-1 silencing on the
proliferation of TC71 and CHLA9 ESFT cells by cell counting. We found that BMI-
1 knockdown significantly inhibited cell growth in confluent ESFT cultures despite
having no affect on logarithmic cell growth (Figure 13D). Our data suggests that
cell growth inhibition at high cell density in BMI-1 knockdown ESFT cells is likely
attributed to activation of cell contact inhibition.
49
Figure 13. Knockdown of BMI-1 in ESFT restores contact inhibition. A. Western blot analysis
confirming BMI-1 knockdown in TC71 and CHLA9 ESFT cells transduced with retroviral non-BMI-
1 silencing control vector (pSuperNS) and BMI-1 shRNA expression vector (pSuperBMI-1). B. 4x
(inset, top left) and 10x Phase contrast images of logarithmic, confluent, and post confluent
cultures of TC71 and CHLA9 ESFT cells stably expressing the control (pSuperNS) and the BMI-1
shRNA vectors (pSuperBMI-1).
50
Figure 13 continued. Knockdown of BMI-1 in ESFT restores contact inhibition. C&D. While BMI-
1 silencing had no affect on logarithmic growth of TC71 and CHLA9 ESFT cells, BMI-1
knockdown TC71 and CHLA cells fail to proliferate in post- confluent culture as assessed by cell
counting. Number of viable cells counted in biological triplicate over a period of 5-6 days at low
cell density and high cell density on 6-well tissue culture plates. The starting cell number for low
cell density count is 100,000-200,000 cells and the initial cell number for high cell density count is
1.5-2.0 million cells.
Loss of BMI-1 induced death and cell cycle arrest in high cell-density cultures
To evaluate whether the apparent growth arrest phenotype was due to an
increase in cell death or a result of cell cycle arrest, we quantified the percentage
of viable BMI-1 knockdown ESFT cells by trypan blue exclusion assay and
analyzed their cell cycle profiles by PI staining followed by analysis on a flow
cytometer. We found that knockdown of BMI-1 did not induce cell death in low-
51
density cultures (Figure 14A). In contrast, at high cell density, we found that BMI-
1 silencing resulted in a significant decrease in cell viability (Figure 14A). This
observation verifies the role of BMI-1 in sustaining ESFT cell viability in post-
confluent cultures. Analysis of the cell cycle profiles of TC71 and CHLA9 BMI-1
knockdown ESFT cells showed a similar conclusion. As shown in figure 14B,
both control and BMI-1 knockdown cells exhibited cell cycle profiles representing
healthy proliferating ESFT cells at low cell density. At high cell density, however,
we observed a distinct cell cycle profile between the control and the BMI-1
knockdown cells. Notably, TC71 BMI-1 knockdown cells underwent G1 cell cycle
arrest upon reaching confluence, as evidenced by an increase in the percentage
of G1 cell population and a decrease in the percentage of S phase cell
population, comparing to non-BMI-1-silencing control cells (Figure 14C). Over
time in post-confluent cultures, the majority of TC71 BMI-1 knockdown cells
succumbed to death as evidenced by a significant increase in the percentage of
cells in sub-G1 phase (data not shown). Similarly, in CHLA9 ESFT cells, we also
made the same observation that silencing of BMI-1 led to an accumulation cells
in Sub-G1 phase, which is indicative of cell death, in high-cell density cultures.
(Figure 14C). This suggests that although G1 cell cycle arrest precedes death in
TC71 BMI-1 knockdown cells, loss of BMI-1 in cells grown at high-cell density will
eventually lead to cell death despite cellular context. Moreover, G1/S cell cycle
progression and transition is dependent on the expression and activity of several
key cell cycle regulatory proteins including CDK4, CDK2, CYCLIN E and CYCLIN
52
D1(Collins et al., 1997). We therefore evaluated the expression level of CDK4,
CDK2, CYCLIN E, and CYCLIN D1 proteins at high cell density over time in both
TC71 and CHLA9 BMI-1 knockdown ESFT cells. As illustrated in Figure 14D,
CDK4, CDK2, CYCLIN E, and CYCLIN D1 proteins were all significantly down-
regulated over time in both TC71 and CHLA9 BMI-1 knockdown ESFT cells
comparing to non-BMI-1-silencing control cells. This intriguing observation
implicates that cell cycle progression was likely halted in the absence of high
levels of BMI-1 expression in high-cell density ESFT cultures. Thus, in two
genetically unique ESFT cell lines, we found that BMI-1 mediates density
dependent inhibition of cell death and cell cycle progression. Future study will
aim to identify the precise molecular mechanism underlying BMI-1-associated
cell cycle defect and death in these BMI-1 knockdown ESFT cells.
53
Figure 14. Loss of BMI-1 induces death and cell cycle arrest. A. Trypan-blue cell viability assay
shows a decrease in the percentage of viable TC71 and CHLA9 BMI-1 knockdown (pSuperBMI-
1) cells comparing to control (pSuperNS) cells in high-cell density cultures. B&C. Cell cycle
analysis of CHLA9 and TC71 control and BMI-1 knockdown cells at high and low cell density.
DNA content of ESFT cells grown under high-cell density condition has been analyzed by FACS
4-5 days post-seeding. Cell cycle analysis of ESFT cells grown under low-cell density condition
has been conducted on cells 2 days post-seeding. Cell cycle profile has been modeled and
analyzed using Flow-Jo software. Estimated percentage±SD of G1, S, G2/M, and Sub-G1 are
indicated below the cell cycle plots. D. Western blot analysis of cell cycle regulatory proteins at
low cell density (thin bar) and high cell density (thick bar) shows a significant decrease in their
expression over time at high cell density. Protein lysates have been harvested on the days
indicated.
54
Figure 14 continued
55
Figure 14 continued
56
Loss of BMI-1 destabilized YAP oncoprotein, a downstream effector of cell-
contact inhibition.
The Hippo tumor suppressor pathway is highly conserved in both
Drosophila and mammals (Zhao et al., 2008). The signaling cascade culminates
in destabilization and inactivation of Yorkie (Drosophila)/YAP(mammals).
Emerging data have shown that mutations in components of the Hippo pathway
contribute to loss of cell contact inhibition in cancer (Zhao et al., 2008). In
particular, YAP inactivation by the Hippo pathway is required to suppress contact
inhibition of cell growth. Mechanistically, growth at high cell density can lead to
phosphorylation, cytoplasmic retention, and subsequent degradation of YAP in
normal cells (Zhao et al., 2010; Zhao et al., 2007). In our study, we hypothesize
that destabilization of YAP, the effector of the Hippo tumor suppressor pathway,
is involved in cell contact inhibition in BMI-1 knockdown cells. To this end, we
investigated the expression level of YAP in BMI-1 knockdown cells in comparison
to the control cells. Intriguingly, in both BMI-1 knockdown TC71 and CHLA9
ESFT cells, YAP protein expression gradually decreased over time at high cell
density (Figure 15A), consistent with our hypothesis that YAP is inactivated and
degraded in confluent BMI-1 knockdown ESFT cells. However, we did not
observe a significant difference in YAP cytoplasmic localization between BMI-1
knockdown and control cells (data not shown). This raises the possibility that
BMI-1 may help stabilize YAP by protecting it from degradation rather than
nuclear export. Noteworthy, endogenous BMI-1 protein expression was down-
57
regulated in BMI-1 knockdown cells over time at confluence (Figure 15A),
suggesting a possible existence of a feed-back loop regulating BMI-1 expression
or its protein stability in response to cell contact inhibition.
Finally, it remains possible that a decrease in YAP protein expression in
BMI-1 knockdown ESFT cells could be attributed to a decrease in its transcription
at high cell density. To test this possibility, we performed QRT-PCR analysis to
quantify YAP1 transcript level in TC71 and CHLA9 ESFT cells. As shown in
Figure 15B, we found that at high cell density, YAP1 transcript was not
significantly altered in BMI-1 knockdown cells compared to the control. This leads
us to believe that mechanisms involved in post-translational modification of YAP
may play a key role in regulating its subsequent degradation.
58
Figure 15. Loss of BMI-1 destablizes YAP oncoprotein, a downstream effector of cell-contact
inhibition. A. Time course western blot analysis of YAP protein level shows a decrease in YAP
protein expression in TC71 and CHLA9 BMI-1 knockdown (pSuperBMI-1) comparing to control
(pSuperNS) cells at high cell density. Protein lysates analyzed were collected from low cell
density (thin bar) and high cell density (thick bar) ESFT cultures. B. QRT-PCR analysis of YAP1
transcripts does not show a significant difference between TC71/CHLA9 control (pSuperNS) and
BMI-1 knockdown (pSuperBMI-1) ESFT cells at low and high cell density.
59
Figure 15 continued
60
YAP functions as an oncogene in Ewing’s Sarcoma.
Deregulation of YAP can render cells refractory to cell contact inhibition
resulting in tumor outgrowth (Zhao et al., 2007). As described earlier in this
study, loss of BMI-1 in ESFT cells at high cell density resulted in growth arrest
and cell death in association with YAP destabilization. This led us to speculate
whether YAP might function as an oncogene in ESFT. To determine if YAP plays
a critical role in promoting ESFT tumorgenicity, we used two different shRNA
oligos against YAP and monitored the growth of these YAP knockdown ESFT
cells over time (Figure 16A). In both TC71 and CHLA9 ESFT cells, YAP
knockdown significantly decreased their proliferation and viability (Figure 16B
and 16C). Importantly, knockdown of YAP in TC71 and CHLA9 cells impaired
their ability to form colonies in soft agar, an in vitro measure of tumorigenicity
(Figure 16D). Finally, to establish whether YAP might promote tumorigenicity
more globally in ESFT, we induced YAP knockdown in two additional ESFT cell
lines, A4573 and A673. Consistent with our observation in TC71 and CHLA9
ESFT cells, the colony forming ability of both A673 and A4573 ESFT cells was
also significantly diminished as a result of YAP silencing (data not shown).
Together, these data lend compelling evidence to support the oncogenic function
of YAP in this family of aggressive tumors.
61
Figure 16. YAP functions as an oncogene in ESFT. A. Western blot analysis confirming YAP
protein knockdown in TC71 and CHLA9 ESFT cells transduced with two different shRNAs against
YAP (shYAP#1 and #2) and a control shRNA (shNS). B. Proliferation analysis of YAP knockdown
TC71 and CHLA9 ESFT cells by cell counting over a period of 7 or 9 days shows a dramatic
decrease in cell growth as a result of YAP loss (Error bar= SEM). o =shYAP#1; x= shYAP#2;
=shNS C. Trypan blue exclusion assay shows a significant decrease YAP knockdown TC71 and
CHLA9 ESFT cell viability. (Error bar= SEM) D. Soft agar assay confirms YAP knockdown inhibits
anchorage independent growth of TC71 (Seeding cell number =5000 cells) and CHLA9 (Seeding
cell number =20,000 cells) ESFT cells. Representative image of one of the three biological
replicates is shown above.
62
Figure 16 continued
63
Chapter 3 Discussion
Our previous study has provided compelling evidence to support BMI-1’s
role as an oncogene in ESFT in a CDKN2A independent manner (Douglas et al.,
2008). In this study, we explored the role of BMI-1 in mediating other hallmarks of
cancer, namely, loss of cell contact inhibition, through a novel CDKN2A-
independent mechanism. Cell contact inhibition is a phenomenon whereby
normal cells cease to proliferate upon achieving confluency (Abercrombie and
Ambrose, 1958; Abercrombie and Heaysman, 1954). Most cancer cells are
insensitive to mechanisms regulating cell contact inhibition proliferation and
movement, resulting in uncontrolled proliferation and aggressive invasion into
neighboring tissues (Abercrombie, 1979). Many signaling pathways have been
implicated in mediating cell contact inhibition. The connection between the
polycomb group protein BMI-1 and cell contact inhibition has not yet been
elucidated. Here, we have provided compelling evidence to support the role of
BMI-1 in suppressing contact inhibition of cell growth in two independent ESFT
cells with distinct CDKN2A and TP53 status. We have shown a similar lack of
contact inhibited phenotype in these two ESFT cell lines despite their genotypic
differences, suggesting that the mechanism underlying contact inhibition is partly
independent of CDKN2A and TP53 function. Although the precise mechanism
underlying BMI-1-mediated suppression of contact inhibition remains enigmatic,
we believe that high level of BMI-1 expression confers ESFT cells a growth
advantage, thereby sets a stage for the development of a more aggressive
64
tumorigenic phenotype. In contrast, loss of BMI-1 expression in ESFT cells
restores their ability to censor their own growth/microenvironment much like
normal cells, consistent with the notion that BMI-1 silencing increases cancer
cells sensitivity to anti-tumor drugs (Alchanati et al., 2009; Qin et al., 2008).
Therefore, loss of BMI-1 can make a cancer cell more vulnerable to anti-
proliferative signaling.
Interestingly, loss of BMI-1 has been linked to premature differentiation of
multipotent stem/progenitor cells (Oguro et al.; Pietersen et al., 2008). Contact
inhibition has been implicated to be a characteristic feature of more differentiated
cell types (Lim et al., 1981; Rodesch, 1973). Thus, it is conceivable that loss of
BMI-1 in highly undifferentiated ESFT cells increases their differentiation state by
limiting their proliferative capacity and tumorigenicity through activation of cell
signaling pathways regulating contact inhibition.
ESFT cell lines are derived from aggressive ESFT primary tumors from
different individuals with different genotype and phenotypic characteristics. From
our experience, p16
INK4A
and p53 mutant cell lines such as TC71 used in this
study grow much more rapidly and are more tumorigenic compared to p16
INK4A
and p53 wild-type CHLA9 cell line. Armed with intact p16
INK4A
and p53 tumor
suppressors, it is conceivable that CHLA9 cell line can be more sensitive to
contact inhibition than TC71 ESFT cells. Our study shows that TC71 BMI-1
knockdown cells undergo G1 cell cycle arrest before they succumb to death at
high cell density. Unlike TC71 cells, death is the overwhelming phenotype we
65
have observed in CHLA9 BMI-1 knockdown cells in high cell density cultures. We
reason that the difference in the sequence of biological events that happened
after cells reached confluence is either due to technical reasons or the genetic
difference between the two cell lines. Nonetheless, our study suggests that BMI-
1 mediated density-dependent promotion of growth is not entirely dependent on
the p16
INK4A
and/or p53 status of the cell lines. Another molecular mechanism is
likely involved in mediating density-dependent cell cycle arrest and death in BMI-
1 knockdown ESFT cells.
The YAP oncogene, discovered as a part of the human chromosome
11p22 amplicon, is highly expressed in a number of human cancers. (Zhao et al.,
2008). The YAP oncoprotein functions as a transcription coactivator and a key
regulator of organ size and contact inhibition downstream of the Hippo pathway
(Zeng and Hong, 2008). In mediating cell contact inhibition, YAP is
phosphorylated and inactivated by the Hippo pathway kinase cascade, which
leads to its destabilization and degradation in the cytoplasm (Zhao et al., 2010;
Zhao et al., 2007). In this study, we showed that at high cell density, YAP
oncoprotein is significantly destabilized and degraded in ESFT cells only in the
presence of BMI-1 knockdown. This suggests that BMI-1 and YAP may interact
and reinforce each other’s pro-survival function. In addition to its oncogenic role,
other groups have shown that YAP may function as a tumor suppressor in a
different cellular context (Basu et al., 2003; Bertini et al., 2009; Lapi et al., 2008).
We find that YAP functions as a growth-promoting oncogene in ESFT and its loss
66
leads to significant decrease in ESFT survival and anchorage-independent
growth. Together, our studies suggest that tumor cell survival at high cell density
requires the cooperative pro-survival, pro-malignant function of both BMI-1 and
YAP. Cells that have escaped contact inhibition are highly likely to form
malignant foci and advanced tumors that can invade neighboring tissues and
metastasize to distant sites (Abercrombie, 1979; Eagle and Levine, 1967).
The molecular mechanism connecting the polycomb group protein BMI-1
and YAP remain a mystery. One study on Drosophila sensory neuron
development reveals that mutations in polycomb group genes phenocopied
defects in the components of Hippo pathway and that polycomb group proteins
physically interact with the components of the hippo signaling cascade (Parrish et
al., 2007). Thus, it is possible that BMI-1 may interact with upstream members of
the hippo pathway that negatively regulate YAP stability. BMI-1 functions as a
component of a large polycomb complex, termed polycomb repressive complex I
(PRC1). PRC1 in association with another polycomb repressive complex II
(PRC2) together coordinately alter chromatin structure and mediate gene
silencing by tri-methylating histone 3 on lysine 27 and ubiquitinating histone H2A
on lysine 119 (Bracken and Helin, 2009). It remains a question whether BMI-1 by
itself or components of PRC1 can physically associate with YAP, thereby
influencing its post-translational modification state and stabilizing its function in
the nucleus.
67
The molecular component responsible for sensing cell-to-cell contact and
destabilizing YAP oncoprotein is unclear. A few cytoskeleton-related proteins,
including Merlin (Mer), Expanded (Ex) and Kibra, are implicated directly or
indirectly to inhibit YAP (Yorkie in Drosophila). (Badouel et al., 2009;
Baumgartner et al.; Genevet et al.; Hamaratoglu et al., 2006; Striedinger et al.,
2008; Yu et al.). There is also evidence to suggest that direct cell-to-cell contact
and the integrity of adherens junctions have tremendous impact on YAP stability
(Nishioka et al., 2009). Interestingly, induction of merlin expression in
schwannoma cells inhibited their growth only in confluent cultures, reminiscent of
the growth phenotype of BMI-1 knockdown ESFT cells at confluence (Morrison et
al., 2001). It is thus conceivable that merlin and other cytoskeletal proteins may
be involved in mediating cell contact inhibition upstream of YAP in BMI-1
knockdown ESFT cells. Moreover, gene expression profiling of ESFT cells from
our previous study shows that cell-to-cell and cell-to-matrix adhesion to be the
major biological processes affected as a result of BMI-1 knockdown (Douglas et
al., 2008). It will be worth to investigate if any of the adhesion genes identified as
a result of BMI-1 knockdown may mediate cell contact inhibition in conjunction
with the hippo pathway converging onto YAP.
In summary, our study provides compelling evidence to suggest that BMI-
1 plays a pivotal role in suppressing cell density dependent growth via stabilizing
YAP. Our discovery suggests that YAP is a cooperative oncogene in ESFT and a
critical effector of BMI-1-mediated cell contact inhibition of growth. Future studies
68
will aim to further shed light on the relationship between BMI-1 and the
components of the hippo signaling pathway culminating to YAP. Understanding
the molecular mechanism underlying hallmarks of cancer will enable us to
develop more effective therapeutic strategy against ESFT as well as other highly
aggressive tumor.
69
Chapter 4
EWS-FLI1 and BMI-1 cooperate in Ewing’s sarcoma tumorigenesis
Chapter 4 Introduction
Ewing’s Sarcoma Family of Tumor (ESFT) is a heterogeneous group of
soft tissue or bone tumor affecting mostly children or young adults. Although
ESFTs are histologically defined as undifferentiated, small, round blue cells, the
origins of ESFT pathogenesis remains obscure. Due to its rarity and lack of
primary anatomical site of occurrence, others and we have come to believe that
ESFT may arise from rare migratory stem cells (Meltzer, 2007). Indeed, recent
data suggests ESFTs may arise from neural crest (Cavazzana et al., 1987; Coles
et al., 2008; von Levetzow et al., 2010; Wahl et al., 2010) or mesenchymal
progenitor stem cells (Castillero-Trejo et al., 2005; Miyagawa et al., 2008; Riggi
et al., 2005; Riggi et al., 2008; Tirode et al., 2007).
The molecular basis of ESFT is beginning to be elucidated. Cytogenetic
analyses have identified the t(11;22)(q24;q12) chromosomal translocation in
approximately 85-90% of ESFT cases (Turc-Carel et al., 1988). The resulting
gene rearrangement creates a chimeric gene EWS-FLI1, consisting of the N-
terminal trans-activation domain of the TET family EWS (EWSR1) gene on
chromosome 22 and the C-terminal DNA binding domain of an ETS transcription
factor family gene FLI1 on chromosome 11. The aberrant transcript encodes a
potent transcription factor, EWS-FLI1(Ordonez et al., 2009). The remaining
cases of ESFT harbor a fusion between EWS and other ETS transcription factor
70
gene family members, including ERG, FEV, ETV1, or E1AF. Other gene
rearrangements between EWS related gene family members such as TLS/FUS
to ETS transcription factor ERG are detected in rare cases (Arvand and Denny,
2001).
EWS-ETS fusions are believed to play a major role in ESFT initiation and
maintenance by acting as dominant oncoproteins. For example, expression of
EWS-FLI1 in NIH3T3 murine fibroblasts can induce oncogenic transformation,
anchorage independent growth, as well as generate tumors in immunodeficient
mice (May et al., 1993; Thompson et al., 1999). Further, ectopic expression of
EWS-FLI1 in murine bone-marrow derived mesenchymal progentor cells also
results in the development of ESFT-like tumor in mice (Castillero-Trejo et al.,
2005; Riggi et al., 2005). However, EWS-FLI1 does not always induce malignant
transformation in target cells. Depending on the cellular context, EWS-FLI1
modulates different biological processes, including cell death (Deneen and
Denny, 2001), growth arrest (Lessnick et al., 2002), and differentiation (Torchia
et al., 2003). Identifying the ESFT cell of origin has been challenging. To date, no
primary human cells can be transformed with the expression of EWS-FLI1 alone.
Therefore, elucidating the cell of origin and identifying additional cooperative
oncogenic lesions will be necessary to shed light on the mechanism of ESFT
pathogenesis.
Genetically engineered mouse models can be very useful tools for studying
the pathogenesis of most cancer. Unfortunately, development of engineered
71
ESFT mouse models has been proven challenging due to cellular toxicity elicited
by ectopic expression of EWS-FLI1. However, recent efforts to create conditional
knock-in EWS-FLI1 mice did not successfully generate mice susceptible to
developing ESFT. Instead, these EWS-FLI1 knock-in mice succumb to myeloid-
erythroid leukemia or poorly differentiated sarcoma in a p53 null setting(Lin et al.,
2008; Torchia et al., 2007). Thus, while a newer version of ESFT transgenic mice
is being conceived and developed, most researchers rely on xenograft model
and cell culture systems to gain insights on the pathogensis of ESFT.
Experiments using heterologous cell systems and ESFT cell lines have
helped identify several candidate EWS-FLI1 target genes. On one hand, MYC,
ID2, CCND1, IGF1, EZH2, GLI1, NKX2.2, TOPK, CAV1, CD99, PDGFR, NROB1
and PIM3 are among those genes that are directly or indirectly induced by EWS-
FLI1. On the other, CDKN1A, CDKN1B, CDKN1C, TGFBRII, ZYX and IGFBP3
are directly or indirectly repressed by EWS-FLI1 expression (Ordonez et al.,
2009). By identifying downstream targets and signaling networks of EWS-FLI1,
we are beginning to understand the mechanism underlying EWS-FLI1 driven
transformation.
Transformation of primary human cells into cancer with a single oncogene
has been proven to be impossible (Hahn and Weinberg, 2002). Work of Hahn
and Weinberg suggests that multiple cooperative hits are necessary for cellular
transformation. Indeed, expression of EWS-FLI1 oncogene alone in primary
human cells has been shown to induce a p53- dependent growth arrest and not
72
cellular transformation (Lessnick et al., 2002). Inactivation of the p53 tumor
suppressor pathway renders the cells tolerant to EWS-FLI1 expression in primary
human cells, suggesting that p53 mutation cooperates with EWS-FLI1 to drive
ESFT tumorigensis. Paradoxically, the majority of the established primary ESFT
tumors exhibit functional, wild type p53 protein and yet are still able to express
EWS-FLI1 (Huang et al., 2005). This suggests that ESFT cell of origin utilizes
other genetic or epigenetic mechanisms to silence tumor suppressor pathways
during tumorigenesis.
Many molecular factors responsible for stem cell maintenance are
deregulated in cancer. The stem cell self-renewal gene BMI-1 is highly
expressed in many types of cancer including ESFT(Douglas et al., 2008). BMI-1
functions as a component of the polycomb group repressive complex 1 (PRC1).
It mediates gene silencing by modifying chromatin structure. In most stem cells
and cancer, it promotes proliferation by binding to and repress CDKN2A locus,
which encodes tumor suppressors p16
INK4A
and ARF. Silencing of p16
INK4A
results in inactivation of Rb. Repression of ARF leads to inactivation of p53
pathway. Therefore, elevated BMI-1 expression in cells results in uncontrolled
cell cycle progression (Bracken and Helin, 2009).
We hypothesize that BMI-1, a highly expressed protein in ESFT and an
epigenetic regulator of the ARF-p53 pathway, is necessary for cell tolerance of
EWS-FLI1 expression in primary human cells. In this study, we will investigate
the effect of EWS-FLI1 expression in BMI-1 over-expressing primary human
73
fibroblasts (MRC5), which inherently expresses low levels of BMI-1, and
determine if EWS-FLI1 and BMI-1 cooperate to induce malignant transformation
of MRC5 cells.
74
Chapter 4 Results
Ectopic EWS-FLI1 expression induced growth arrest in MRC5 primary human
fibroblasts
Expression of EWS-FLI1 has been shown to induce a p53-dependent
growth arrest in hTERT-immortalized primary human neonatal foreskin
fibroblasts (Lessnick et al., 2002). To verify the previously observed EWS-FLI1-
induced growth phenotype in our cellular system, we ectopically expressed EWS-
FLI1 using an EWS-FLI1 lenti-viral expression vector, pCCL, in MRC5 embryonic
human lung fibroblasts (Figure 17A). As shown in figure 17B, EWS-FLI1-
expressing MRC5 lost their healthy spindle-shape morphology. Proliferation
analysis revealed that EWS-FLI1 dramatically decreased the growth of MRC5
cells (Figure 17C). Together, these results indicate that EWS-FLI1 is just as toxic
to MRC5 fibroblasts as to foreskin fibroblasts.
75
Figure 17. Ectopic EWS-FLI1 expression induces growth arrest in MRC5 primary human
fibroblasts. A. pCCL lenti-viral vector map. V5 tagged EWS-FLI1 is constitutively expressed and
driven by the activity of the MND promoter. Tranduced cells are marked by EGFP positivity. B.
Left pCCL EWS-FLI1 transduced MRC5 cells have lost their fibroblastic spindle-shaped
morphology three days after transduction. Right Confirmation of EWS-FLI1 expression by
western blot three days post-transduction. C. EWS-FLI1-expressing MRC5 cells undergo growth
arrest three days after viral transduction as assessed by MTS proliferation assay. ( Empty
vector; EWS-FLI1 expressing vector)
A.
76
BMI-1 abrogates EWS-FLI1-induced growth arrest in MRC5 cells
To determine if BMI-1 abrogates EWS-FLI1 induced cellular toxicity, we
genetically engineered MRC5 cells to stably express both BMI-1 and EWS-FLI1
using BMI-1-retro-viral (pBabe) and EWS-FLI1-lenti-viral (pCCL) expression
vectors (Figure 18A). As illustrated in figure 18C and 18B, MRC5 cells
expressing only EWS-FLI1 underwent growth arrest and a change in morphology
resembling dying cells. In contrast, BMI-1-expressing MRC5 cells are permissive
for EWS-FLI1 expression. Although initially, MRC5 cells expressing EWS-FLI1
and BMI-1 did not proliferate as well as non-EWS-FLI1-expressing control cells,
these cells were able to recover over time (data not shown). We observed that
after one-month post-EWS-FLI1 viral transduction, BMI1-expressing MRC5 cells
appeared to have lost contact inhibition and continued to proliferate over time
(Figure 18B and C).
77
Figure 18. BMI-1 abrogates EWS-FLI1-induced growth arrest in MRC5 cells. A. Western blot
analysis confirming BMI-1 and EWS-FLI1 expression in MRC5 cells day 3 post-viral transduction.
B. Phase contrast images show EWS-FLI1 induces changes in MRC5 cell morphology
resembling of dying cells, while BMI-1 cooperates with EWS-FLI1 in MRC5s to promote loss of
contact inhibition growth. C. As assessed by MTS proliferation assay, BMI-1 over-expressing
MRC5 fibroblasts continue to grow despite EWS-FLI1 expression one-month post-viral
transduction (Blue Vector control; Red BMI-1 only, Green EWS-FLI1 only, Black, BMI-1 and
EWS-FLI1-expressing MRC5 cells).
78
BMI-1 abrogates EWS-FLI1-induced oncogene-induced senescence in primary
human cells
Oncogene activation in normal cells can induce premature cellular
senescence known as oncogene-induced senescence (OIS)(Serrano et al.,
1997). We speculate whether BMI-1, a suppressor of senescence in stem cells
(Park et al., 2004), may protect MRC5 cells from EWS-FLI1-induced OIS. To test
our hypothesis, we stained MRC5 cells for senescence-associated β-
galactosidase activity (pH 6.0), a marker of senescent cells. As seen in figure 19,
the vast majority of MRC5 cells expressing only EWS-FLI1 stained positive for
senescence associated-β-gal activity in contrast to BMI-1 and EWS-FLI1-
expressing MRC5 cells. Noteworthy, there were very few surviving MRC5 cells
that express only EWS-FLI1, suggesting that EWS-FLI1 expressing MRC5 cells
became senescent and died in the absence of BMI-1 expression (Figure 19).
79
Figure 19. BMI-1 abrogates EWS-FLI1-induced oncogene-induced senescence MRC5 cells.
EWS-FLI1 expressing MRC5 cells in the absence of BMI-1 expression have become senescent.
Senescent cells display positive staining for senescence-associated β-galactosidase activity
(Blue). Only rare β-galactosidase positive cells can be identified in BMI-1 over-expressing cells
that express EWS-FLI1.
To rule out the possibility that clonal evolution could confer a growth
advantage to non-EWS-FLI1-expressing BMI-1-overexpressing cells, we
conducted RT-PCR analysis to ensure our MRC5 cells maintained EWS-FLI1
expression one-month post-viral transduction (We did not have enough protein
lysate or cells to verify the protein expression of EWS-FLI1 in MRC5 cells that
have become senescent). As shown in Figure 20, we found that EWS-FLI1 was
expressed at the transcript level in MRC5 cells one-month after transduction.
However, it will be important to determine if EWS-FLI1 transcript and protein can
80
be maintained at a physiologic level comparable to ESFT in BMI-1-expressing
MRC5 cells over time in culture in future studies.
81
Figure 20. EWS-FLI1 expression is maintained one-month post-transduction. EWS-FLI1
transcript is detected one month post-transduction in MRC5 cells by RT-PCR. TC71 ESFT cDNA
serves as a positive control for EWS-FLI1 PCR. Both MRC5 cDNA and H
2
O are negative
controls.
Development of a doxycycline-inducible EWS-FLI1 expression system in MRC5
cells.
Establishing a stable EWS-FLI1 expression system in primary human cells
was a challenging task. Lenti-viral transduction experiments performed with
equal amount of viral titre varied from one experiment to the other. As a result,
we were not able to establish primary cell lines expressing comparable level of
EWS-FLI1. We found that the level of EWS-FLI1 expression was a critical
determinant of the EWS-FLI1-induced phenotype. On one hand, too much of
EWS-FLI1 expression instantly killed the cells. On the other, we could not detect
any protein expression by immunofluorescence or western blot if too little EWS-
FLI1 was expressed. As a result, we were not able to consistently reproduce the
EWS-FLI1-induced phenotype using the pCCL expression system. Thus, to avoid
any technical and experimental variance attributed by the constitutive lenti-viral
82
expression system (pCCL), we developed a doxycycline-inducible EWS-FLI1
lenti-viral expression vector, pSLIE-hygro (Figure 21). With as little as 10ng/mL of
doxycycline, we could induce a robust level of EWS-FLI1 expression in MRC5
cells (Figure 21B). The pSLIE-hygro system is far from perfect, quantitative RT-
PCR analysis on non-induced MRC5 cells revealed a leaky EWS-FLI1
expression in the absence of doxycycline (Figure 21B and data not shown). This
leakiness in EWS-FLI1 expression is enough to induce some level of cellular
toxicity to MRC5 cells, which could compromise our experimental interpretation
of the results. Future experiments will be required to isolate and expand clones of
non-leaky pSLIE-hygro-transduced MRC5 cells. Nonetheless, the pSLIE-hygro
system allows us to control EWS-FLI1 expression to a level comparable to ESFT
cells. By changing the viral titre and/or the amount of doxycycline used, we could
modulate the amount EWS-FLI1 expressed (Figure 21C). To further validate the
feasibility of the pSLIEhygro vector, we showed that too much EWS-FLI1
expression induced growth arrest in MRC5 cells (100 and 500 ng/mL dox), while
expression of EWS-FLI1 to a physiologic level comparable to ESFT cell
(10ng/mL dox) enabled the cells to remain proliferative (Figure 21D). This may
indicate that MRC5 inherently tolerate EWS-FLI1 expression in contradiction to
what was shown above using the constitutive EWS-FLI1 expression system.
However, our preliminary data does not suggest that BMI-1 does not confer a
growth advantage to EWS-FLI1-expressing primary human cells. We found that
EWS-FLI1 is well tolerated by BMI-1 expressing-MRC5 cells using the inducible
83
EWS-FLI1 system (data not shown). We believe that the proliferative effect of
BMI-1 is required to extend the life span of EWS-FLI1-expressing primary human
fibroblasts so that these cells can acquire additional oncogenic mutations
necessary for malignant transformation. To this end, we are currently isolating
and expanding non-leaky pSLIE-hygro transduced BMI-1 expressing MRC5 cells
to address the question whether BMI-1 functions as a cooperative oncogene in
MRC5 cells.
84
Figure 21. Development of a doxycycline-inducible EWS-FLI1 expression system in MRC5 cells.
A. pSLIEhygro/Venus vector map modified from pSLIK lentiviral vector (Shin et al., 2006). The
vector constitutively expresses a yellow fluorescent protein Venus or hygromycin-resistance
gene. V5 tagged EWS-FLI1 (EFV5) expression can be induced by the addition of doxycycline in
the culture media. B. Western blot analysis confirming EWS-FLI1 protein expression in pSLIE-
hygro transduced MRC5 cells. 10ng/mL of doxycycline is sufficient to induce EWS-FLI1
expression. C. As assessed by quantitative RT-PCR, EWS-FLI1 transcript level is increased in
MRC5 cells treated with increasing amount of doxycycline (10,100, and 500 ng/mL doxycycline).
10ng/mL doxycycline is sufficient to induce EWS-FLI1 transcript level comparable to TC71 and
TC32 ESFT cells. MRC5 cDNA serves as a negative control for EWS-FLI1 PCR. D. Induction of
EWS-FLI1 with 100 and 500 ng/mL of doxycycline can induce growth arrest in MRC5 cells, while
10 ng/mL of doxycycline does not affect proliferation of MRC5 cells as assessed by MTS
proliferation assay.
85
Mechanism of BMI-1-mediated protection against EWS-FLI1-induced OIS
Our preliminary results showed that BMI-1 protects MRC5 cells against
EWS-FLI1-induced OIS (Figure 19). Expression of EWS-FLI1 in heterologous
cell systems have found that inactivation of the p16
INK4A
and p53 tumor
suppressor pathways renders cells tolerant to EWS-FLI1 expression (Deneen
and Denny, 2001; Lessnick et al., 2002). In addition, p16
INK4A
and ARF-p53
pathways have been implicated in OIS in primary human and rodent cells
(Courtois-Cox et al., 2008; Serrano et al., 1997). We therefore hypothesize that
BMI-1 may inactivate OIS by repressing p16
INK4A
and ARF-p53 pathways. Our
preliminary data showed that BMI-1 repressed p16
INK4A
expression in MRC5 cells
(Figure 22). However, EWS-FLI1 expression did not induce further p16
INK4A
up-
regulation above baseline level in these cells. This indicates that BMI-1-mediated
repression of p16
INK4A
unlikely plays a key role in protecting cells from EWS-FLI1-
induced OIS. To determine if BMI-1 repressed ARF-p53 pathway, we looked for
evidence to support ARF-p53 repression in MRC5 cells, as judged by the level of
p21 expression. As shown in figure 22, BMI-1 did not repress p21 expression
and p21 protein level was unchanged despite the expression of EWS-FLI1.
Based on this preliminary data, we do not have evidence to indicate that BMI-1-
mediated inactivation of p16
INK4A
or ARF-p53 abrogates EWS-FLI1-induced OIS.
However, as alluded to above, if BMI-1-mediated protection against OIS can only
be seen in long-term cultures, comparing the expression level of effectors
involved in OIS pathway in late passage cultures will be more informative.
86
Moreover, recent review on the mechanism underlying OIS reveals a prevalent
role of DNA damage response in mediating OIS preferentially in human cells
(Evan and d'Adda di Fagagna, 2009). Although BMI-1 is known to repress
p16
INK4A
/ARF (Jacobs et al., 1999a), a novel role of BMI-1 in DNA damage
response pathway has been recently discovered (Liu et al., 2009). It is
conceivable that BMI-1 protects MRC5 cells from EWS-FLI1-induced OIS by
inactivating DNA damage response pathway.
Figure 22. Possible mechanism of BMI-1-mediated protection against EWS-FLI1-induced OIS.
p16
INK4A
and p21 (a downstream effector of ARF-p53 pathway) protein expression are assessed
by western blot in EWS-FLI1 expressing MRC5 cells in the presence or absence of BMI-1 over-
expression day 3 post-viral transduction. p16
INK4A
is repressed by BMI-1; however, EWS-FLI1
does not up-regulate p16
INK4A
expression. Likewise, p21 expression is unchanged upon EWS-
FLI1 expression. p21 expression does not appear to be regulated by BMI-1 in MRC5 cells.
87
EWS-FLI1-BMI-1 signaling axis
Thus far, we have implicated that high levels of BMI-1 expression may
confer a growth advantage to putative ESFT cell of origin. Whether or not BMI-1
expression is an inherent feature of the ESFT cell of origin (i.e. neural crest stem
cell) or induced by EWS-FLI1 during tumor development remains an intriguing
question. Our preliminary study on MRC5 fibroblasts shows that EWS-FLI1 does
not induce BMI-1 protein expression (Figure 22). To evaluate if EWS-FLI1
modulates BMI-1 expression in ESFT cells, we knocked down EWS-FLI1 in
A4573 and TC71 ESFT cells and observed no change in BMI-1 protein
expression (Figure 23). Our preliminary data either suggest that there is no
relationship between EWS-FLI1 and BMI-1 or that EWS-FLI1 regulates BMI-1
expression in a cell context dependent manner. The latter is likely true as data
from our lab have shown that ectopic expression of EWS-FLI1 in neural crest
stem cells induces robust BMI-1 expression (von Levetzow et al., 2010). It is thus
conceivable that EWS-FLI1-mediated up regulation of BMI-1 expression plays a
key role in ESFT tumor initiation in the ESFT cell of origin. As the tumor becomes
more established, other growth-promoting pathway may serve a more critical role
in maintaining BMI-1 expression.
88
Figure 23. EWS-FLI1 does not regulate BMI-1 in ESFT cells. A. Knockdown of EWS-FLI1 does
not lead to down-regulation of BMI-1 in A4573 ESFT cells as assessed by western blot. pSuper
retroviral vector, a non-EWS-FLI1 targeting shRNA vector (shNS), a type 1 EWS-FLI1 targeting
shRNA vector (shNS#2), or a EWS-FLI1 3’UTR targeting shRNA vector (shEWS-FLI1) are
transfected into A4573 ESFT cells bearing a type 3 EWS-FLI1 fusion. Protein lysates are
collected after 48 hours of puromycin (1.5 µg/mL) selection and analyzed by western blot. B.
EWS-FLI1 silencing does not lead to a decrease in BMI-1 expression in TC71 ESFT cells as
assessed by western blot. A non-EWS-FLI1 targeting shRNA (shNS), a type 1 EWS-FLI1 shRNA
targeting vector (shEWS-FLI1) are transfected into TC71 bearing a type 1 EWS-FLI1 fusion.
Protein lysates are process as described above.
hTERT-immortalized MRC5 cells tolerate EWS-FLI1 expression.
The making of cancer cells requires activation of oncogene, inactivation of
tumor suppressors, as well as reactivation of telomerase activity (Hahn and
Weinberg, 2002). High level of telomerase expression is a characteristic feature
of both tumor and stem cells (Cukusic et al., 2008; Kim et al., 1994). Although
BMI-1 can extend the life span of primary fibroblasts, its expression does not
consistently immortalize cells (Itahana et al., 2003; Park et al., 2004). In our
study, BMI-1 expression in EWS-FLI1- expressing MRC5 cells is not sufficient to
89
induce cellular transformation (data not shown). We therefore hypothesize that
activation of telomerase activity in addition to BMI-1 is required to fully transform
EWS-FLI1 expressing primary human cells. To evaluate our hypothesis, we
conducted a preliminary experiment to test if EWS-FLI1 is tolerated in hTERT-
immortalized MRC5 cells. As shown in figure 24A, we had no difficulty
expressing EWS-FLI1 in hTERT immortalized MRC5 cells using the pCCL EWS-
FLI1 lenti-viral vector. Interestingly, ectopic expression of EWS-FLI1 in hTERT
immortalized MRC5 cells did not induce growth arrest (figure 24B). Albeit
growing slower than the control cells, the EWS-FLI1-expressing hTERT-
immortalized-MRC5 cells were healthy and proliferating. Our preliminary data
suggest that hTERT may substitute BMI-1’s role in stabilizing EWS-FLI1
expression in primary human cells. It remains a pressing question whether
hTERT and EWS-FLI1 expression are sufficient to induce transformation or a
combination of hTERT, BMI-1, and EWS-FLI1, are necessary to reprogram
primary human cells into ESFT-like cells. It is possible that additional factors
might be required to fully convert primary human cells into tumorigenic ESFT
cells.
90
Figure 24. hTERT-immortalized MRC5 cells tolerate EWS-FLI1. A. Western blot analysis
confirming EWS-FLI1 protein expression in hTERT-immortalized MRC5 fibroblasts (a gift from Dr.
Gerard Evan, UCSF). B. EWS-FLI1 is tolerated in hTERT-immortalized MRC5 cells. EWS-FLI1-
expressing hTERT MRC5 cells grow slower than the control cells as assessed by MTS
proliferation assay.
91
Chapter 4 Discussion
In this study, we showed that EWS-FLI1 induced growth arrest in
embryonic primary human lung fibroblast, MRC5. We hypothesize that a stem-
like epigenetic environment renders cells permissive for EWS-FLI1 expression.
To assess our hypothesis, we over-expressed BMI-1, a stem cell self-renewal
factor, in MRC5 cells and found that BMI-1 over-expression abrogated EWS-
FLI1-induced cellular toxicity in MRC5 cells. Our study suggests that BMI-1
functions as a cooperative oncogene in EWS-FLI1-driven tumorigenesis by
inhibiting oncogene-induced senescence (OIS).
Cellular senescence is a natural tumor suppressing response elicited by
cells under various types of stress including oncogene activation (Prieur and
Peeper, 2008). For instance, expression of an oncogene such as RAS in normal
primary human cells can induce irreversible premature growth arrest known as
oncogene-induced senescence (OIS)(Serrano et al., 1997). In our study, we
have assessed whether EWS-FLI1, a growth promoting oncogene in ESFT, may
induce OIS in MRC5 cells. We find that the majority of EWS-FLI1 expressing
MRC5 have become senescent. Although the result contradicts previous study
on EWS-FLI1 expressing primary human neonatal foreskin fibroblasts (Lessnick
et al., 2002), the change in senescent phenotype likely reflects a difference in cell
type or technicalities between the two studies. Intriguingly, MRC5 cells
expressing both BMI-1 and EWS-FLI1, albeit growing slower than control cells
92
not expressing EWS-FLI1, are not senescent. This reveals that BMI-1 acts
against OIS and prepares cells to become fully transformed.
Ectopic expression of EWS-FLI1 in heterologous cellular models elicit
diverse cellular responses including cell death and cell cycle arrest (Deneen and
Denny, 2001; Lessnick et al., 2002). The impact of EWS-FLI1 on other cellular
processes including apoptosis and cell cycle remains to be characterized in
MRC5 cells and MRC5 cells over-expressing BMI-1. We expect that BMI-1, as a
key mediator of G1/S progression, will help sustain the growth of EWS-FLI1-
expressing cells and prevent them from undergoing cell cycle arrest. Although
the mechanism underlying the anti-apoptotic function of BMI-1 is unclear, BMI1
has been shown to repress apoptosis via ARF-p53 pathway (Jacobs et al.,
1999b). Other study has indicated that BMI-1 plays a critical role in regulating
cellular sensitivity to apoptotic stimuli (Li et al., 2010). It will not be surprising that
BMI-1 may protect MRC5 cells from EWS-FLI1-induced apoptosis. We have
preliminary data to show that EWS-FLI1 does induce apoptosis three days after
viral transduction equally in both BMI-1-expressing and non-BMI-1 expressing
MRC5 cells (data not shown). Because BMI-1-mediated abrogation of EWS-
FLI1-induced cellular toxicity is only seen in late passage cells, it will be
interesting to evaluate whether or not BMI-1 can protect EWS-FLI1 cells from
undergoing apoptosis in addition to senescence in late passage MRC5 cells.
The molecular mechanism underlying BMI-1 mediated protection against
EWS-FLI1-induced cellular toxicity has yet to be explored. Clues revealed from
93
previous studies on EWS-FLI1 expressing human foreskin fibroblasts and mouse
embryonic fibroblasts implicate ARF-p53 and/or p16
INK4A
-Rb pathways(Deneen
and Denny, 2001; Lessnick et al., 2002). Activation of OIS has been shown to
result in the accumulation of p53 and p16
INK4A
tumor suppressors (Serrano et al.,
1997). BMI-1 is known to protect stem cells and fibroblasts from replicative
senescence partly by repressing p16
INK4A
-Rb and/or ARF-p53 pathways (Park et
al., 2004). Together, these studies suggest that BMI-1-mediated protection
against EWS-FLI-1-induced oncogenic insult is likely via repressing p16
INK4A
-Rb
and/or ARF-p53 pathways. However, it remains possible that EWS-FLI1 induced
OIS may be dependent on activation of an alternative pathway, the DNA damage
response pathway. Intriguingly, BMI-1 has been implicated in DNA damage
response pathway and DNA damage response has been suggested to be the
default pathway activated in human cells in response to OIS (Evan and d'Adda di
Fagagna, 2009; Liu et al., 2009). Thus, it will worthwhile to dissect the role of
BMI-1 in DNA damage response pathway in response to EWS-FLI1-induced OIS
in addition to p16
INK4A
-Rb and/or ARF-p53 pathways.
Our study shows that BMI-1 over-expression significantly extends the
lifespan of EWS-FLI1 expressing MRC5 cells. However, these cells are not
sufficiently transformed to grow anchorage-independently, an in vitro assessment
of tumorigenicity (data not shown). Hahn and Weinberg et al proposed that the
making of a tumor from primary cells is a multi-step process requiring the
activation of oncogenes, inactivation of tumor suppressors, and maintenance of
94
telomeres. Specifically, they concluded that alterations in RAS, pRB, p53, and
hTERT expression are keys to cellular transformation as they are responsible for
many key biological phenotypes of cancer (Hahn and Weinberg, 2002). It follows
that expression of other key oncogenic elements involved in cellular
transformation such as hTERT and RAS may be necessary to initiate ESFT
pathogenesis in MRC5 cells. However, although hTERT is highly expressed in
ESFT and is an EWS-FLI1 target, there is so far no evidence to implicate RAS to
be deregulated in ESFT primary tumors (Fuchs et al., 2004; Radig et al., 1998).
Preliminary study from our lab shows that hTERT can rescue EWS-FLI1-induced
cellular toxicity much alike BMI-1. It is plausible that EWS-FLI1, BMI-1, and
hTERT together will be sufficient to induce ESFT tumorigeneis. It is also possible
that additional oncogenic factor will be necessary to fully reprogram MRC5 cells
into ESFT-like cells.
It remains enigmatic how BMI-1 expression has become highly elevated in
ESFT cells. One possibility is that higher BMI-1 expression is an inherent feature
of the ESFT cell of origin. This implies that the ESFT cell of origin is a stem cell,
possibly a neural-crest stem cell, which expresses high levels of BMI-1 and is
capable of self-newal. Indeed, ectopic expression of EWS-FLI1 in human
embryonic stem cell derived neural crest stem cells and human mesenchymal
stem cells, which express BMI-1, tolerate EWS-FLI1 expression (Miyagawa et
al., 2008; Riggi et al., 2008; von Levetzow et al., 2010). The other possibility is
that BMI-1 expression is induced as a result of EWS-FLI1 expression in the cell
95
of origin. Data from lab shows that EWS-FLI1 induces BMI-1 expression neural
crest stem cells, a putative ESFT origin (von Levetzow et al., 2010). However,
shRNA and siRNA mediated knockdown of EWS-FLI1 in ESFT cell lines does
not result in a decrease in BMI-1 protein expression. Together, these data imply
that modulation of BMI-1 by EWS-FLI1 is cell context dependent and may occur
only during tumor initiation. Whether or not EWS-FLI1 directly or indirectly target
BMI-1 remains a puzzle. It is also conceivable that deregulation of BMI-1
expression is a result of subsequent oncogenic mutation irrespective to the EWS-
FLI1 status. For example, clonal evolution may have selected for highly
proliferative ESFT cells that express elevated levels of BMI-1. Thus far, we have
compelling evidence to support the role of BMI-1 in ESFT maintenance (Douglas
et al., 2008). Many questions remain pertaining to its involvement in ESFT
initiation. Ideally, it would be more informative to have an ESFT mouse model
designed to recapitulate all the initial oncogenic events during the development
of ESFT. However, despite lack of a good in vivo ESFT model, our study in
primary human cells has implicated a role of BMI-1 in EWS-FLI1-driven
tumorigenesis. More experiments will be conducted using the inducible EWS-
FLI1 system to verify the EWS-FLI1-induced growth phenotype and fully
characterize the mechanism underlying BMI-1-mediated protection against EWS-
FLI1-induced oncogenic insult in primary human cells.
96
Chapter 5
From bench to bedside: a new hope for BMI-1-driven ESFT therapies
The development of effective therapy against ESFT has been challenging
largely due to lack of understanding of its underlying origin and pathogenesis.
Although multimodal treatment has improved the survival of patients with
localized disease, patients diagnosed with metastatic disease still fare poorly
(Balamuth and Womer, 2010). Further, ESFT patients receiving high dose of
cytotoxic chemothereapy are at risk of developing secondary malignancy (Bacci
et al., 2005; Paulussen et al., 2001). Currently, new therapies against the
characteristic ESFT fusion protein, EWS-FLI1, and its cooperative targets have
been in development in the laboratory (Balamuth and Womer, 2010). Efforts from
several research laboratories including ours have used microarray studies to
identify ESFT gene expression signatures in patients in hope to better
understand the progression of the disease (Ohali et al., 2004; Schaefer et al.,
2008; Scotlandi et al., 2009).
Studies from our laboratory have implicated that deregulation of the stem
cell self-renewal factor, BMI-1, plays a key role in promoting ESFT progression.
We have shown that BMI-1 expression is responsible for sustaining ESFT in vitro
and in vivo tumorigenic phenotype including anchorage independent growth, cell
contact inhibition, as well as tumor engraftment and development in NOD-SCID
mice. We have elucidated novel roles of BMI-1 in mediating cell adhesion and
implicated NID1 and YAP to function cooperatively downstream of BMI-1, serving
97
as important effectors of BMI-1-mediated cancerous phenotype. Together, our
studies have identified BMI-1 as a potential therapeutic target and prognostic
marker in treating ESFT.
Targeting BMI-1 therapeutically can be a challenging task due to its
impact on global gene expression. However, as we are beginning to forge its
connection to specific molecular targets downstream, we will be able to design
drugs to more effectively target molecular effectors involved in executing BMI-1-
mediated oncogenic signaling. For example, our studies have identified YAP to
be a potent cooperative oncogene downstream of BMI-1 in ESFT. Multiple
signaling networks including the hedgehog pathway, AKT pathway and the Hippo
pathway converge onto YAP (Basu et al., 2003; Fernandez et al., 2009; Zeng
and Hong, 2008). Thus, inhibiting YAP function will help impede BMI-1-mediated
growth signaling and reduce ESFT tumorigenicity.
Intriguingly, studies on ESFT primary tumors have shown that while the
majority of the tumor samples are BMI-1 positive, up to 25% of the tumors do not
express BMI-1 (Douglas et al., 2008). Survival analysis on ESFT patients reveals
that BMI-1 does not serve as a significant prognostic marker (van Doorninck et
al., 2010). However, molecular studies and gene expression profiling on ESFT
primary tumors suggests that BMI-1 positive and BMI-1 negative tumors differ
significantly in regards to their differentiation state and their mode of
pathogenesis (van Doorninck et al., 2010). Importantly, data from our lab shows
that loss of BMI-1 increases the sensitivity of ESFT cells to IGF-1R antibody,
98
which has been recently reported to have anti-tumor effect against ESFT in a
phase 1 trial. (Olmos et al., 2010; van Doorninck et al., 2010). Thus, BMI-1 may
serve as a useful predictor of drug sensitivity. Understanding the role of BMI-1 in
ESFT pathogenesis will greatly help designing new therapeutic regiment that
could improve the survival rate of ESFT patients.
99
Chapter 6
Material and methods
Cell lines and tumor specimens.
ESFT cell lines, A4573, TC71, TC32, TC252, CHLA9, were obtained from
Dr. Timothy Triche (Children’s Hospital Los Angeles, Los Angeles, CA). ESFT
cell lines, StaET8.2 and StaET7.2, were provided by Dr. Heinrich Kovar
(CCRI,
St. Anna Kinderkrebsforschung, Vienna, Austria). All ESFT cell lines were
propagated in RPMI medium supplemented with 10% fetal bovine serum, L-
glutamine and penicillin/streptomycin (Cellgro Mediatech). MRC5, Tera-2, and
MCF-7 cells were
obtained from American Type Culture Collection (ATCC). H9
human
embryonic stem cells were from Wicell. Human MSCs were obtained from
Tulane Center for Gene Therapy (New Orleans, LA). These cells were
propagated according to the manufacturers’ protocol.
Primary tumor RNA and
tissue slides were obtained from Childrens
Hospital Los Angeles
(CHLA) and additional RNA samples and ESFT tissue
microarrays
were obtained from the Cooperative Human Tissue Network in
Columbus,
OH. All primary tissue samples were anonymized and acquired
in
accordance with approval from the CHLA Committee for Clinical
Investigation.
RNA Isolation and Quantitative real-time reverse transcriptase PCR.
RNA was extracted and DNase I treated using RNAeasy kit. (Qiagen).
cDNA was generated
from DNase I–treated RNA using iScript cDNA synthesis
kit(Bio-Rad). Quantitative
real-time PCR (qRT-PCR) was performed
using
100
validated TaqMan Gene Expression Assays (Applied Biosystems).
Assays were
performed in triplicate on an Applied Biosystems
7900HT Fast real-time PCR
system. Average Ct values were normalized
relative to expression of ACTIN
and/or GAPDH in the same sample.
Expression data was collected from a
minimum of three biological replicate
experiments. For assessment of EWS-FLI1
expression, qRT-PCR was performed using iTaq SYBR Green supermix with
ROX (BIORAD) using the following primer pairs:
5’-CGACTAGTTATGATCAGAGCAGT-3’ (ESBP1)
5’-CCGTTGCTCTGTATTCTTACTGA-3’ (ESBP2)
Western blot.
Cells were lysed in RIPA buffer (150 mM sodium chloride, 1% triton X,
0.5% sodium deoxycholate, 0.1% SDS, 50mM Tris buffer pH 8) containing
protease and phosphatase inhibitor cocktail tablets (Roche). Whole cell lysates
were quantified using DC protein assay (Biorad) and separated by SDS-PAGE
followed by transfer onto a PVDF membrane (Invitrogen). Membrane was
blocked with 5% milk or BSA in TBST for 1h at room temperature before
incubating with primary and secondary antibody. After incubation with secondary
antibody, the blot is immersed with ECL and visualized on X-ray films (Kodak).
Immunohistochemistry
Histological sections and
tissue microarrays were stained with
Hematoxylin and Eosin and anti–BMI-1 antibody (1:50) according to the
BenchMark IHC/ISH staining protocol. Tissue microarrays were digitally scanned
101
and images acquired by the Children's Oncology Group Biopathology
Center
(Columbus, OH) using Aperio ImageScope Scanner and software
(Aperio).
Antibodies
Anti-BMI-1 (clone F6; 1:1000) was obtained from Millipore/Upstate. Anti-
ACTIN (I-19; 1:1000), anti-cMYC (N262; 1:1000), anti-MEL-18 (H-115; 1:1000),
anti-NIDOGEN (H-200; 1:500), p16(C-20; 1:1000), anti-YAP (63.7; 1:1000), anti-
CDK2 (D-12; 1:1000), and Anti-Mouse-HRP(1:2000) were acquired from Santa
Cruz Biotechnology. Anti-CASPASE 3 (1:1000), anti-CYCLIN D1(1:1000), anti-
p21WAF1/CIP1 (1:1000), anti-CDK4 (1:1000), anti-EZH2 (AC22; 1:1000), anti-
GAPDH (14C10; 1:1000), anti-PARP (1:1000), anti-phospho-RB (Ser807/811;
1:1000), Anti-RB (4H1; 1:1000), anti-phospho-YAP (Ser127; 1:1000) were
purchased from Cell Signaling. Anti-FLI1 (1:500) was obtained from US
Biological. Anti-Rabbit HRP (1:10,000) was obtained from Bio-Source. CYCLIIN
E (1:1000) was acquired from BD Biosciences. Anti-V5 (1:1000) antibody was
purchased from Invitrogen.
Senescence-associated β-galactocidase staining (SA-β-gal)
SA-β-gal staining was performed according to Dimri et al. (Dimri et al.,
1995). Briefly, cells were washed with PBS and fixed in 2% formaldehyde/0.2
glutaraldehyde (or 3% formaldehyde) for 3-5 minutes. Fixed cells were washed
with PBS and incubated with SA-β-gal staining solution containing 1 mg/mL 5-
bromo-5-chloro-3-indolyl β-D-galactoside (X-gal), 40mM citric acid/sodium
phosphate, pH 6.0, 5mM potassium ferrocyanide, 5 mM potassium ferricyanide,
102
150mM NaCl, 2mM MgCl
2
. Cells were visualized and images were captured
under a microscope the next day.
Wnt5A conditioned medium
The Wnt5A conditioned medium was produced in collaboration with Mr.
Daniel Wai from Dr. Timothy Triche’s lab. Mouse L Wnt5A and control cells
(ATCC) were cultured in ESFT growth medium for 4 days. The conditioned
medium was removed from cell culture and passed through a sterile filter. The
medium stock was stored frozen until use.
Promoter DNA Methylation analysis by Illumina GoldenGate platform
DNA was isolated from ESFT cells using DNeasy Blood & Tissue kit
(Qiagen). Bisulfite conversion of DNA and methylation assay were performed in
collaboration with Dr. Peter Laird and his team. The protocol for preparing DNA
samples for Illumina GoldenGate assay was previously described (Bibikova et
al., 2006). The degree of methylation was assessed based on the β value. A β
value >0.17 signifies a significant difference in DNA methylation between
samples (Bibikova and Fan, 2009).
Gene knockdown and over-expression.
To knockdown BMI-1, cell
lines were transfected with 50 nmol/L of BMI-1–
targeted
(siBMI1-A, 5'-CCGUCUUAAUUUUCCAUUG-3'; or siBMI1-B, 5'-
GCGGUAACCACCAAUCUUC-3'),
or negative control (siNS) Small interfering
RNA (siRNA) oligonucleotides
(Ambion). Knockdown of NID1 was achieved by
transfection of
100 nmol/L of pre-validated siRNA oligonucleotides (Ambion).
For
103
stable BMI-1 knockdown, siBMI1-A and siBMI1-B DNA oligonucleotide
sequences were cloned into the pSuper-retro-puro short-hairpin
vector backbone
(shBMI1-A and shBMI1-B; Oligoengine). For stable EWS-FLI1 knockdown
against the type I fusion protein, shEF30 (pSuper) vector was generously
provided by Dr. Heinrich Kovar (CCRI, St. Anna Kinderkrebsforschung, Vienna,
Austria). For stable EWS-FLI1 knockdown against 3’UTR of EWS-FLI1,
shEF3’UTR (5’-ATAGACCTGGGAAGCTTAT-3’) was cloned into the pSuper-
retro-puro short-hairpin vector backbone (Smith et al., 2006). A nonsilencing
sequence (5'-ACGCATGCATGCTTGCTTT-3') was similarly cloned to
act as a
negative control (Baek et al., 2005). For gain-of-function studies,
full-length
human BMI-1 cDNA was PCR-amplified from TC-71 ESFT cells,
sequence-
verified, and cloned into pBabe-Puro. Retroviral supernatants
were produced
through tri-transfection of 293FT packaging cells
(Invitrogen) with the appropriate
retroviral construct along
with the packaging plasmids pHit 60 (MLV gag-pol) and
pHit 456
(VSV envelope; provided by Dr. C. Lutzko, CHLA). Viral supernatants
were collected after 48 h for transduction of ESFT cells followed
by selection in 2
µg/mL of puromycin.
The cloning of EWS-FLI1 into pCCL and pSLIK lentiviral vectors was
conducted by our technical staff, Aaron Cooper. The coding sequence of EWS-
FLI1 was PCR amplified from pcDNA/TO-EF, a generous gift from Dr. Timothy
Triche (Children’s Hospital Los Angeles, CA), with a simultaneous deletion of the
normal stop codon. This product was inserted into pENTR/D-TOPO (Invitrogen)
104
and the EWS-FLI1 element was then moved into pLenti4/TO/V5-DEST
(Invitrogen) by LR recombination. EWS-FLI1-V5, hereafter EFV5, was PCR-
amplified from pLenti4/TO/EFV5-DEST with primers containing an EcoRI site
upstream and an MfeI site downstream of the ORF. The EcoRI/MfeI-digested
PCR product was ligated into EcoRI-digested pCCL-MND-X-IRES-eGFP
backbone, a generous gift from Dr. Carolyn Lutzko (Children’s Hospital Los
Angeles, CA). For cloning of EWS-FLI1-V5 into pSLIK vector, EWS-FLI1-V5 was
amplified from pLenti4/TO/EFV5-DEST with primers adding NheI sites on either
end. The product was inserted between the SpeI and XbaI sites of pEN_TmiRc3
(ATCC # MBA-248). The TRE-EFV5 element was then moved into pSLIK-Hygro
(Single Lentivector for Inducible Knockdown, ATCC # MBA-237) and pSLIK-
Venus (ATCC # MBA-234) by LR recombination, yielding pSLIE-EFV5-Hygro
(Single Lentivector for Inducible Expression) and pSLIE-EFV5-Venus.
To knockdown YAP in ESFT cells, the following vectors were obtained
from Sigma-Aldrich:
1. Non-targeting shRNA control vector pLKO puro.1 shNS (5’→3’)
(CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTT
GTTTTT)
2. YAP1-targetting pLKO puro.1 shYAP #1 (5’→3’)
(CCGGGCCACCAAGCTAGATAAAGAACTCGAGTTCTTTATCTAGCTTGGTGG
CTTTTTG)
105
3. YAP1-targeting pLKO puro.1 shYAP #2 (5’→3’)
(CCGGCCCAGTTAAATGTTCACCAATCTCGAGATTGGTGAACATTTAACTGG
GTTTTTG)
Lenti-virus packaging and transduction were performed following standard
protocols. Briefly, Lenti-viral supernatant was produced by cotransfecting the
vector plasmid with helper plasmids pCD/NL-BH (gag-pol; a gift from Dr. Gregor
von Levetzow) and pMD2.G (VSV-G env; Addgene) into 293T cells. Viral
supernatant was collected 48h post-transfection.
Assessment of cell growth, adhesion, and tumorigenicity.
Transfected/transduced
cells were plated in triplicate wells and total/viable
cell counts
and cell viability were determined using a ViCell XR cell counter
(Beckman Coulter). For cell growth experiments associated with cell contact
inhibition, all cells were maintained in RPMI media supplemented to 5% FBS, L-
glutamine and penicillin/streptomycin (Cellgro Mediatech).
MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium) proliferation assay was performed using CellTiter 96
Aqueous One Solution (Promega) following manufacturer’s instruction.
Absorbance at 495 nm was measured using GeniosPro plate reader (Tecan).
Average value of at least 3 replicates was calculated and the values were
normalized to the optical density measured at day 1 of the experiment (20 h after
106
plating). 500-1000 cells were seeded in each well on a 96 well plate
supplemented with 100 µL of growth medium.
Flow cytometry studies of
apoptosis were completed following standard
protocols for Annexin
V detection and DNA content using a FACScan instrument
(Becton Dickinson). Acquired data was analyzed using CellQuest software. For
cell cycle analysis, cells were harvested and fixed with ice-cold 70% ethanol
overnight at 4°C. Fixed cells were treated with 100 µg/ml RNase A stained with
50µg/ml propidium iodide. DNA content of fixed cells was analyzed using a
FACScan instrument (BD). Cell cycle data were processed and analyzed using
FlowJo software (Treestar).
For the study of anchorage-independent growth,
transduced cells were
plated as single cell suspensions in 0.35%
noble agar (Difco) in Iscove's medium
supplemented with 20%
fetal bovine serum, 2 µg/mL of puromycin, 2 mmol/L of
L-glutamine, 100 IU of penicillin, and 100 mg/mL of streptomycin.
Cellular layers
were sandwiched between feeder layers consisting
of 0.7% noble agar in
Iscove's medium supplemented with 10%
fetal bovine serum, 2 µg/mL of
puromycin, 2 mmol/L of
L-glutamine, 100 IU of penicillin, and 100 mg/mL of
streptomycin. Cells were fed weekly with 0.35% noble agar containing growth
described above.
In vitro cell adhesion assays were performed as described (Weeraratna et
al., 2002).
Briefly, 5 x 10
4
cells/well were plated in 96-well plates and
then
washed with PBS and fixed at 30-min intervals. Adherent
cells were stained with
107
0.2% crystal violet followed by several
washes with water. Crystal violet was
extracted with 2% SDS
and the absorbance (570 nm) of each well was measured
using
a GeniosPro plate reader (Tecan).
In vivo tumorigenicity studies were performed
with the assurance of the
Institutional Animal Care and Usage
Committee. Non-obese diabetic (NOD)-
severe combined immunodeficiency
(SCID) mice (Charles River Laboratories)
were injected s.c.
with 5 x 10
6
tumor cells and tumor volume was measured using
calipers. Mice were sacrificed when tumors reached a size of
1.5 cm in any
dimension.
DNA isolation and Genomic real-time PCR determination of p16 status.
Genomic real-time
PCR was performed as previously described (Labuhn
et al., 2001) and calculation
of CDKN2A exon 1a gene copy number determined
relative to GAPDH
copy number in each sample (Berggren et al., 2003).
Microarray analysis.
RNA was extracted, DNase I–treated,
and purified (RNeasy Minikit;
Qiagen) from A4573 cells 48 h
post-siRNA transfection. Samples were prepared
in the CHLA genome
core using the GeneChip whole transcript sense target
labeling
assay manual (Affymetrix) and hybridization to HuEx1.0 microarrays
was
carried out following the manufacturer's instructions. Cell
intensity file data were
quantile-normalized and summarized
using the iterative probe logarithmic
intensity error estimation
(iterPLIER) within the Affymetrix expression console
software.
A linear model fit was determined for each transcript using
the LIMMA
108
package (linear models for microarray data; (Smyth, 2004))
and differentially
expressed genes between control and BMI-1
knockdown cells identified on the
basis of statistically significant
differences in transcript level signal intensity (P <
0.01)
and a fold change of 1.5. Metacore
4
(GeneGo, Inc.) analytical
tools were
used to identify functional gene ontology categories.
Statistics
Student t-test was utilized to compare samples between two different
categories. p<0.05 was considered significant.
109
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Appendix
Peer-reviewed publication in Cancer Research (2008)
BMI-1 promotes Ewing sarcoma tumorigenicity independent
of CDKN2A repression
125
BMI-1 Promotes Ewing Sarcoma Tumorigenicity Independent
ofCDKN2A Repression
Dorothea Douglas,
1
Jessie Hao-Ru Hsu,
1
Long Hung,
1
Aaron Cooper,
1
Diana Abdueva,
2
John van Doorninck,
1
Grace Peng,
1
Hiro Shimada,
3
Timothy J. Triche,
2,3
and Elizabeth R. Lawlor
1,2,3
1
Division of Hematology-Oncology, Department of Pediatrics, Childrens Hospital Los Angeles, and Departments of
2
Pediatrics and
3
Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California
Abstract
Deregulation of the polycomb group gene BMI-1 is implicated
in the pathogenesis of many human cancers. In this study, we
have investigated if the Ewing sarcoma family of tumors
(ESFT) expresses BMI-1 and whether it functions as an
oncogene in this highly aggressive group of bone and soft
tissuetumors.OurdatashowthatBMI-1ishighlyexpressedby
ESFT cells and that, although it does not significantly affect
proliferation or survival, BMI-1 actively promotes anchorage-
independent growth in vitro and tumorigenicity in vivo.
Moreover, we find that BMI-1 promotes the tumorigenicity
of both p16 wild-type and p16-null cell lines, demonstrating
thatthemechanismofBMI-1oncogenicfunctioninESFTis,at
least in part, independent of CDKN2A repression. Expression
profiling studies of ESFT cells following BMI-1 knockdown
reveal that BMI-1 regulates the expression of hundreds of
downstream target genes including, in particular, genes
involved in both differentiation and development as well as
cell-cell and cell-matrix adhesion. Gain and loss of function
assays confirm that BMI-1 represses the expression of the
adhesion-associated basement membrane protein nidogen 1.
Inaddition,althoughBMI-1promotesESFTadhesion,nidogen
1 inhibits cellular adhesion in vitro.Together,these data
support a pivotal role for BMI-1 ESFT pathogenesis and
suggest that its oncogenic function in these tumors is in part
mediated through modulation of adhesion pathways. [Cancer
Res 2008;68(16):6507–15]
Introduction
Members of the Ewing sarcoma family of tumors (ESFT) are
characterizedbytheexpressionofchimericfusiononcogenes,most
commonly EWS-FLI1 (reviewed in ref. 1). EWS-FLI1 transforms
NIH-3T3 fibroblasts and its knockdown in ESFT cells dramatically
inhibits tumorigenicity (2). In contrast, EWS-FLI1 induces a p53-
dependent cell cycle arrest in primary human fibroblasts and loss
of p16 is required for the transformation of primary murine
fibroblasts (reviewed in ref. 1). Appropriate suppression of innate
tumor suppressor pathways is, therefore, necessary for EWS-FLI1–
mediated malignant transformation. Unfortunately, evaluation of
primary ESFT samples has, thus far, yielded little insight into the
mechanism of this inactivation in vivo.Only25%ofESFTcases
exhibit either mutation of p53 or deletion of the p16
INK4a
/ARF
locus, and although these patients exhibit a worse prognosis, they
clearly represent a minority of cases (3).
Polycomb group family proteins are central players in the
maintenance of stem cell self-renewal and pluripotency as well as
in the control of cellular differentiation and development (4–6).
Polycomb proteins assemble into large complexes, termed PRC1
and PRC2, to repress transcription through modulation of the
chromatin structure and their expression is frequently deregulated
in cancer (7–9). The PRC1 gene bmi-1 was first identified as an
oncogene that collaborates with c-myc in a murine model of
lymphomagenesis (10). Studies of bmi-1 knockout mice later
showed that BMI-1 regulates the self-renewal of hematopoietic,
neural, and neural crest stem cells (reviewed in ref. 9).
Mechanistically, BMI-1 maintains stemness and prevents prema-
ture cellular senescence in large part through transcriptional
repression of Cdkn2a (11). The CDKN2A locus encodes p16
INK4a
and p14
ARF
,genes that contribute to cell cycle regulation and
apoptosis through modulation of the retinoblastoma and p53
pathways. In somatic stem cells, BMI-1 functionally inhibits both
pathways thereby supporting self-renewal and immortality. Down-
regulation of BMI-1 expression during cellular differentiation is
associated with the release of this inhibition.
Inadditiontoc-myc (10,12),bmi-1 cooperateswiththeleukemia-
associated translocations E2a-Pbx1 (13) and Hoxa9-Meis1 (14) in
murineleukemogenesis,andithasalsobeenimplicatedintheorigin
of nasopharyngeal carcinoma (15), neuroblastoma (16, 17), and
medulloblastoma (18, 19). Moreover, the EWS-FLI1–related protein
TLS-ERGcanimmortalizeprimaryhumanhematopoieticstemcells
that have up-regulated endogenous BMI-1 expression (20). Finally,
the self-renewal and tumorigenicity of leukemia, neuroblastoma,
and breast cancer stem cells has been linked to BMI-1 function
(14, 17, 21). Thus, BMI-1 is expressed by and functions as an
oncogene in many types of human cancer. Importantly, although
directtranscriptionalrepressionoftheCDKN2A locuscontributesto
the oncogenic activity of BMI-1 in cellular models (12), recent
studies have suggested that other mechanisms of BMI-1–mediated
tumor promotion exist and thatthese are functionally independent
of CDKN2A repression (19, 22–24).
In this study, we have investigated whether BMI-1 functions as
an oncogene in ESFT. Our findings confirm that BMI-1 is highly
expressed by ESFT cells and that it promotes anchorage-
independent growth in vitro and tumor formation in vivo.
Furthermore, our data also show that the mechanism of BMI-1–
mediated tumorigenicity in ESFTis, at least in part, independent of
CDKN2A repression and that BMI-1 regulates pathways involved in
cell differentiation and development as well as cell adhesion. In
particular, these data supportthe hypothesis that BMI-1 expression
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
D. Douglas and J.H-R. Hsu contributed equally to this work.
Requests for reprints: Elizabeth R. Lawlor, Childrens Hospital Los Angeles, 4650
Sunset Boulevard, Los Angeles, CA 90027. Phone: 323-361-8579; Fax: 323-361-4902;
E-mail: elawlor@chla.usc.edu.
I2008 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-07-6152
www.aacrjournals.org 6507 Cancer Res 2008; 68: (16). August 15, 2008
Research Article
126
is central to the pathogenesis of ESFTand that modulation of cell
adhesion pathways contributes to BMI-1–mediated tumorigenicity
in this tumor family.
Materials and Methods
Cell lines and tumor specimens. ESFT cell lines obtained from Dr.
Timothy Triche (Los Angeles, CA) and Dr. Heinrich Kovar (CCRI, St. Anna
Kinderkrebsforschung, Vienna, Austria) were grown as described (25, 26).
MRC5, Tera-2, and MCF-7 cells were obtained from American Type Culture
Collection, and H9 human embryonic stem cells were from Wicell. Primary
tumor RNA and tissue slides were obtained from Childrens Hospital Los
Angeles (CHLA) and additional RNA samples and ESFT tissue microarrays
were obtained from the Cooperative Human Tissue Network in Columbus,
OH. All primary tissue samples were anonymized and acquired in accor-
dance with approval from the CHLA Committee for Clinical Investigation.
Quantitative real-time reverse transcriptase PCR. cDNA was
generated from DNase I–treated RNA (iScript; Bio-Rad) and quantitative
real-time reverse transcriptase PCR (QRT-PCR) was performed using
validated TaqMan Gene Expression Assays (Applied Biosystems). Assays
were performed in triplicate on an Applied Biosystems 7900HT Fast real-
time PCR system and average Ct values normalized relative to expression of
ACTIN and/or GAPDH in the same sample. Expression data was collected
from a minimum of three replicate experiments.
Western blot and immunohistochemistry. Western blots of whole cell
lysates were performed using the following primary antibodies at 1:1,000
dilutions: BMI-1 (Millipore); PARP, phosphoretinoblastoma, total retino-
blastoma, and p21 (Cell Signaling); p16, p14ARF, and ACTIN (Santa Cruz
Biotechnology). Histologic sections and tissue microarrays were stained
with anti–BMI-1 antibody (1:5,000; Millipore). Tissue microarrays were
digitally scanned and images acquired by the Children’s Oncology Group
Biopathology Center (Columbus, OH) using Aperio ImageScope Scanner
and software (Aperio).
Gene knockdown and overexpression. To knockdown BMI-1,celllines
were transfected with 50 nmol/L of BMI-1–targeted (siBMI1-A, 5¶-CCGU-
CUUAAUUUUCCAUUG-3¶; or siBMI1-B, 5¶-GCGGUAACCACCAAUCUUC-3¶),
or negative control (siNS) Small interfering RNA (siRNA) oligonucleotides
(Ambion).KnockdownofNID1 wasachievedbytransfectionof100nmol/Lof
prevalidatedsiRNAoligonucleotides(Ambion).ForstableBMI-1 knockdown,
siBMI1-AandsiBMI1-BDNAoligonucleotidesequenceswereclonedintothe
pSuper-retro-puro short-hairpin vectorbackbone (shBMI1-Aand shBMI1-B;
Oligoengine). A nonsilencing sequence (5¶-ACGCATGCATGCTTGCTTT-3¶)
was similarly cloned to act as a negative control (27). For gain-of-function
studies,full-lengthhumanBMI-1 cDNAwasPCR-amplifiedfromTC-71cells,
sequence-verified,andclonedintopBabe-Puro.Retroviralsupernatantswere
produced through tritransfection of 293FTpackaging cells (Invitrogen) with
theappropriateretroviral constructalongwiththe packaging plasmidspHit
60 (MLV gag-pol) and pHit 456 (VSV envelope; provided by Dr. C. Lutzko,
CHLA).Viralsupernatantswerecollectedafter48hfortransductionofESFT
cells followed by selection in 2 Ag/mL of puromycin.
Assessment of cell growth, adhesion, and tumorigenicity. Trans-
fected/transduced cells were plated in triplicate wells and total cell counts
and cell viability determined using a ViCell XR cell counter (Beckman
Coulter). Flowcytometrystudies of cell cycleandapoptosiswere completed
following standard protocolsforAnnexinV detectionand DNAcontent (28)
using a FACScan instrument (Becton Dickinson). For the study of
anchorage-independent growth, transduced cells were plated as single cell
suspensions in 0.35% noble agar (Difco) in Iscove’s medium supplemented
with 20% fetal bovine serum, 2 Ag/mL of puromycin, 2 mmol/L of
L-glutamine, 100 IU of penicillin, and 100 mg/mL of streptomycin. Cellular
layers were sandwiched between feeder layers consisting of 0.7% noble agar
in Iscove’s medium supplemented with 10% fetal bovine serum, 2 Ag/mL of
puromycin, 2 mmol/L of L-glutamine, 100 IU of penicillin, and 100 mg/mL
of streptomycin. In vitro cell adhesion assays were performed as described
(29). Briefly, 5 ! 10
4
cells/well were plated in 96-well plates and then
washed with PBS and fixed at 30-min intervals. Adherent cells were stained
with0.2% crystal violet followedby several washeswithwater. Crystalviolet
was extracted with 2% SDS and the absorbance (570 nm) of each well was
measured using a GeniosPro plate reader (Tecan). In vivo studies were
performed with the assurance of the Institutional Animal Care and Usage
Committee. Nonobese diabetic (NOD)-severe combined immunodeficiency
(SCID) mice (Charles River Laboratories) were injected s.c. with 5 ! 10
6
tumor cells and tumor volume was measured using calipers. Mice were
sacrificed when tumors reached a size ofz1.5 cm in any dimension.
Genomic real-time PCR determination of p16 status. Genomic real-
time PCR was performed as previously described (30) and calculation of
CDKN2A exon 1a gene copy number determined relative to GAPDH copy
number in each sample (31).
Microarray analysis. RNA was extracted, DNase I–treated, and purified
(RNeasy Minikit; Qiagen) from A4573 cells 48 h post-siRNA transfection.
Samples were prepared in the CHLA genome core using the GeneChip
whole transcript sense target labeling assay manual (Affymetrix) and
hybridization to HuEx1.0 microarrays was carried out following the
manufacturer’s instructions. Cell intensity file data were quantile-normal-
ized and summarized using the iterative probe logarithmic intensity error
estimation (iterPLIER) within the Affymetrix expression console software. A
linear model fit was determined for each transcript using the LIMMA
package (linear models for microarray data; ref. 32) and differentially
expressed genes between control and BMI-1 knockdown cells identified on
the basis of statistically significant differences in transcript level signal
intensity (P < 0.01) and a fold change of z1.5. Metacore
4
(GeneGo, Inc.)
analytical tools were used to identify functional gene ontology categories.
Results
BMI-1 is expressed by Ewing tumors. Expression microarray
data generated from primary tumors and ESFT cell lines were
analyzed for BMI-1 expression. As shown in Fig. 1A, BMI-1 was
variablebutdetectableinallsamples.Thesedatawerecorroborated
by QRT-PCR analysis of an independent cohort of primary tumors
and cell lines (Fig. 1B). Nontransformed control cells, consisting of
primary human fibroblasts (MRC5), bone marrow–derived mesen-
chymal stromal cells (MSC), and human embryonic stem cells
(hESC), were similarly evaluated. To ensure equivalent extracellular
environmental stimuli, all cells were collected during logarithmic
growth phase and, with the exception of the hESC, were grown in
the same medium (RPMI with 10% fetal bovine serum) for 24 hours
prior to harvesting. Western blot (Fig. 1C)andimmunohistochem-
ical analysis (Fig. 1D)confirmedBMI-1proteinexpressioninESFT
cell lines and primary tumors, respectively. Histologic evaluation of
67 ESFTtumorbiopsies revealed BMI-1 to be diffusely and robustly
expressedbytumorcellsin49cases(73%),whereasendothelialcells
and infiltrating lymphocytes were negative for the protein (Fig. 1D,
case I and II). In 18 cases (27%), only rare and/or very weakly
stainedBMI-1–positive tumorcellsweredetected(Fig.1D, case III).
In summary, using both RNA and protein studies, we have found
that BMI-1 is highly expressed by the vast majority of ESFT cells.
We are currently investigating the potential clinical and/or
biological significance of high versus low-level BMI-1 expression
in primary ESFT.
BMI-1 knockdown does not induce ESFT cell death or
CDKN2A expression. It has been previously shown that BMI-1
promotes the proliferation of normal human fibroblasts (33), and
that loss of BMI-1 in multiple human cancer cell lines induces cell
death (34). To determine if BMI-1 promotes the survival and/or
proliferation of ESFTcells, we assessed the effects of altered BMI-1
expression on ESFT cells grown in standard culture conditions.
Two different siRNA sequences were used and both sequences
4
http://www.genego.com/
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127
effectively repressed BMI-1 in ESFT and in MCF-7 breast cancer
cells (Fig. 2A). In contrast to MCF-7 cells, in which BMI-1
knockdown was found to significantly inhibit both proliferation
and survival, BMI-1 knockdown had no effect on the growth of
ESFT cells (Fig. 2B; Supplementary Data Fig. S1). In corroboration
with these findings, we also found that whereas overexpression of
BMI-1 promoted the proliferation of MRC5 fibroblasts, it had no
effect on ESFT cell proliferation (data not shown).
Because BMI-1 has been reported to exert its effects through the
repressionofCDKN2A anditsproteinproductsp16andp14ARF,we
reasoned that the lack of effect of BMI-1 modulation on ESFT cell
proliferation and death may be a consequence of CDKN2A deletion
inthecelllines.UsingacombinationofQRT-PCRandWesternblot,
we found that A4573, StaET8.2, and StaET7.2 express the CDKN2A
transcript and both A4573 and StaET8.2 express the p16 protein
(Supplementary Data Fig. S2A and B). Genomic real-time PCR (30)
revealedthatTC71,TC32,andTC252cellsallhavehomozygousloss
ofCDKN2A,StatET8.2,andStatET7.2arewild-typeforthelocus,and
A4573 is hemizygously deleted (Supplementary Data Fig. S2C and
D). Thus, BMI-1 knockdown does not inhibit ESFT cell growth
irrespective of their p16 status. Next, we set out to determine
whether BMI-1 represses CDKN2A transcript expression in p16+
ESFTcells. Unexpectedly, we found that although BMI-1 repressed
the expression of CDKN2A/p16 in human MSC and Tera2
embryonal carcinoma cells, no significant or consistent effect was
observedinESFTcelllines(SupplementaryDataFig.S3A,B,andC).
The alternate protein product encoded by the CDKN2A locus,
p14ARF, was not expressed to detectable levels by ESFT (data not
shown). BMI-1–dependent repression of a p21-retinoblastoma
pathway has recently been implicated in the control of embryonic
neural stem cell self-renewal (35). To determine if BMI-1 may be
targeting p21 rather than p16 in ESFT cells, we also evaluated
CDKN1A expression and p21 protein levels following BMI-1 gain
and loss of function. p21 protein levels were unaffected by BMI-1
knockdown in all six ESFT cell lines tested whereas CDKN1A was
up-regulated in A4573 cells and down-regulated in StaET8.2 cells
(datanot shown). Nochange inpRB-phosphorylationwasobserved
in any ESFT cell line following BMI-1 knockdown (Supplementary
Data Fig. S3D). Together, these data indicate that BMI-1 does not
significantly modulate either CDKN2A or CDKN1A expression in
ESFTandthefailureofBMI-1topromoteESFTproliferationand/or
survival is not a consequence of CDKN2A/p16 loss.
Figure 1. Ewing tumors (ESFT)expressBMI-1. A, gene expression profiling using Affymetrix U133A GeneChips detects expression of BMI-1 (Pcgf-4; Probeset
ID 202265_at) in 94 primary ESFT and 10 ESFT cell lines. Dashed lines, median expression levels; bars, interquantile ranges. B, QRT-PCR of 20 primary ESFT
and6ESFTcelllinesconfirmsdetectablebutvariableexpressionofBMI-1 inESFT.ExpressioninnonmalignantH9humanembryonicstemcells(hESC),fourprimary
humanbonemarrow–derivedmesenchymalstromalcellcultures(MSC1–MSC4),andMRC5lungembryofibroblasts isuniformlylow.Expressionlevelswererepeated
in triplicate and normalized relative to the median expression of GAPDH and ACTIN in the same sample. C, Western blot analysis detects BMI-1 protein in ESFT
cell lines whereas expression in normal fibroblasts (MRC5) and MSC is very low to undetectable. The variability of protein expression in both ESFT and nonmalignant
cell lines correlates with transcript expression in B. D, immunohistochemical analysis of 67 ESFT biopsy samples reveals that in 49 cases, tumor cells are highly
positive for BMI-1 (e.g., case I and case II). Unlike tumor cells, endothelial cells in case I (arrow) and infiltrating lymphocytes in case II (circled)arenegative.
In 18 cases (e.g., case III), BMI-1 is weakly expressed or is only expressed by rare cells.
BMI-1 Promotes Ewing Sarcoma Tumorigenicity
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128
The mechanism of BMI-1–mediated repression of p16 has been
shown to involve direct binding of the CDKN2A promoter and to
depend on the presence of a functional retinoblastoma protein
(pRB; ref. 36). ESFT cells display hyperphosphorylated (inactive)
pRB when grown in standard culture conditions and although
serumwithdrawalleadstoareductioninpRBphosphorylation,this
is accompanied by a reduction in total pRB expression (Supple-
mentaryDataFig.S3E).TodetermineifBMI-1knockdownisableto
effect changes in ESFTgrowth in conditions in which pRB may be
relatively more active, we repeated cell growth assays in serum-free
conditions. Consistent with our earlier findings, we observed no
significant effects of BMI-1 knockdown on CDKN2A expression
level,cellproliferation,orcelldeath(Fig.2C andD;datanotshown).
BMI-1 promotes the anchorage-independent growth and
tumorigenicity of Ewing tumor cells. Although BMI-1 loss did
not alter ESFTproliferation or death, we observed that knockdown
dramatically altered the morphologic characteristics of A4573
cells in vitro. Whereas these cells normally grow as three-
dimensional clusters, knockdown of BMI-1 reproducibly resulted
in conversion to growth as adherent cellular monolayers (Fig. 3A).
We therefore reasoned that BMI-1 might contribute to anchorage-
independent colony formation. To assess whether the anchorage-
independent growth of ESFT cells in vitro is affected by altered
BMI-1 expression levels, we performed soft agar assays of cells that
were genetically modified to express altered levels of BMI-1. As
shown, knockdown of BMI-1 inhibited colony formation (Fig. 3B),
Figure 2. BMI-1 knockdown does not affect ESFT cell proliferation or death. A, QRT-PCR confirms the knockdown of BMI-1 following transfection of siRNA
oligonucleotides (siBMI1A and siBMI1B)comparedwithmockorcontrol(siNS) transfected cells. ESFT cell lines—A4573, StaET8.2, TC71, and MCF7: positive
controls, breast cancer cell line. RNA was harvested from cells 48 h posttransfection and gene expression normalized to GAPDH expression levels in the same
sample. Columns, averages of three replicate experiments; bars, SD. B, siRNA-mediated knockdown of BMI-1 significantly inhibits growth of MCF7 but not ESFT
cells. Cells transfected with siRNA oligonucleotides were counted daily for 4 days and the number of viable cells plotted. BMI-1 knockdown (siBMI1)countswere
comparedateachtimepointtocontrol(siNS)transfectedcells.Points, averagecountsofthreereplicateexperiments;bars, SD.DataisshownforsiBMI1B.Equivalent
results were obtained for the siBMI1A sequence (data not shown). C, QRT-PCR analysis shows no effect of BMI-1 knockdown on CDKN2A expression in serum-free
culture. Stably transduced A4573 cells (shBMI1 and shNS control) were grown in serum-free conditions for 48 h prior to RNA isolation. Columns, average of
replicate experiments with gene expression expressed relative to GAPDH in the same samples; bars, SD. D, knockdown of BMI-1 has no effect on ESFT cell
proliferation or death in cells grown in serum-free conditions. A4573 cells stably transduced with a BMI-1 hairpin construct (shBMI1)andgrowninserum-free
conditions for 48 h show no change in cell death (left; fluorescence-activated cell sorting analysis of Annexin-V/propidium iodide–stained cells) or cell proliferation
(right; fluorescence-activated cell sorting analysis of fixed, propidium iodide–stained cells).
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129
whereas overexpression of BMI-1 led to a more rapid formation of
macroscopic colonies and an increase in macroscopic colony
number (Fig. 3C). Confirmation of the specificity of the effects of
BMI-1 knockdown was achieved by demonstrating equivalent
effectsusinga second shRNAsequence(SupplementaryDataFig. S4).
To determine if BMI-1 promotion of ESFT colony formation
in vitro correlates with in vivo tumorigenicity, we evaluated xeno-
graft tumor formation in NOD-SCID mice. As shown in Fig. 4A and
B,therateofengraftmentofTC71tumorcellsdirectlycorrelated
with BMI-1 expression levels. For shBMI1 cells, the median time
to measurable tumor was 16 days compared with only 13 days for
shNS cells (P <0.05).In contrast, BMI-1–overexpressingcells
formed tumors within 10.5 days compared with 15 days for empty
vector–transduced cells (P = 0.005). Similarly, whereas A4573 shNS
control cells formed tumors with a median time of 15 days, only
one of five shBMI1-recipient mice developed a tumor and this did
not appear until 37 days postimplantation (data not shown). To
confirm that the xenografts did not form from cells that had
escaped genetic modification, variable expression of BMI-1 was
confirmed in the excised tumors (Fig. 4C).
BMI-1 knockdown effects significant changes in develop-
mental and cell adhesion pathways. Having established that
BMI-1 promotes ESFT tumorigenicity in the absence of CDKN2A
modulation,weinitiated studiestoidentifynoveleffectorsofBMI-1
action. To achieve this, we performed expression profiling of A4573
cellsfollowingacuteknockdownofBMI-1andfoundthatnearly900
known genes were significantly affected (P<0.01),and245ofthese
were altered at least 2-fold (Supplementary Table S1A; GEO
accession series no. GSE12064). Assessment of the gene ontology
designations of these BMI-1–responsive genes found several path-
ways to be significantly overrepresented, in particular, pathways
involved in cellular development as well as adhesion and invasion
(Table1).Inaddition,whereasmanygenesintheseoverrepresented
pathways were induced by BMI-1 knockdown (e.g., NOTCH1,
WNT5A, TIMP2,and TIMP3), others were down-regulated (e.g.,
COL5A2, COL1A2,and ICAM2)demonstratingthatalthoughBMI-1
is known to function as a transcriptional repressor, inhibition of its
expression does not exclusively lead to gene induction and genes
thatareindirectlyregulatedbyBMI-1,eitherup-regulated ordown-
regulated, may contribute to its function as an oncogene.
In an attempt to identify the downstream genes that are most
likely to mediate tumorigenicity, we compared our data to a
previously published analysis of BMI-1 knockdown in DAOY
medulloblastoma cells (19). Raw data from this study was extracted
from the National Center for Biotechnology Information GEO
database (series no. GSE7578) and processed using the same
methodology used for analysis of our ESFT data (described in
Materials and Methods). Comparison of the two independent gene
lists reveals that expression of 101 genes was significantly and
commonly altered by BMI-1 knockdown in both A4573 and DAOY
cells (Supplementary Table S1B). Importantly, whereas A4573 cells
express p16, DAOYare p16-null, further supporting the designation
of these BMI-1 targets as p16-independent (37). Moreover, DAOY
cells were studied following shRNA transduction and antibiotic
selection, whereas A4573 cells were analyzed following acute
siRNA-mediated knockdown. Thus, the common effects on gene
expression cannot be attributed to the consequences of experi-
mental design. Rather, these 101 genes are likely to represent true
Figure 3. BMI-1 promotes the anchorage-independent growth of ESFT cells. A, phase contrast images of A4573 cells growing in standard culture conditions.
siRNA-mediated knockdown of BMI-1 (siBMI1)causesA4573cellstoflattenwhereasLipofectamine-treatedandcontroltransfected(siNS)cellscontinuetogrowas
three-dimensional clusters. B, stable knockdown of BMI-1 (shBMI1B) impairs colony formation of ESFT cells in soft agar. Images are of macroscopic colonies on
the day at which control cell (shNS) media was depleted for each of the three respective cell lines. Each well is representative of six replicate wells for each condition.
Similar results were obtained with a second BMI-1–targeted shRNA construct (see Supplementary Data Fig. S4). C, ESFT cells transduced to overexpress BMI-1
(pBp-BMI1) have a reduced time to macroscopic colony formation and increased colony number in soft agar when compared with control, empty vector–transduced
cells (pBp-V). Images are of macroscopic colonies on the day at which pBp-BMI1 media was depleted for each of the three respective cell lines. Each well is
representative of six replicate wells for each condition.
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130
BMI-1 targets. A high degree of overlap in overrepresented
biological processes between the two model systems is also
observed, with the same developmental and cell adhesion genes
featuring prominently(Table1).Toensurethattheoverlapbetween
the gene sets was not the result of chance, we computed the
probability of obtaining the observed number of overlaps under a
hypergeometric distribution and confirmed the degree of overlap
to be highly statistically significant (P < 0.001).
In view of the striking overrepresentation of genes involved in
cellular adhesion, we evaluated the consequences of BMI-1
modulation on the adhesion of A4573 cells in vitro. As shown in
Fig. 5A,adhesion is promoted by BMI-1 overexpression and
inhibited by BMI-1 knockdown in these cells. To validate that
adhesion genes identified by microarray analysis are bona fide
targets of BMI-1, we performed QRT-PCR and Western blot to
assess the expression levels of NID1 and VEZT,andtheirrespective
protein products, nidogen 1 and vezatin. Although expression of
both genes was consistently and reproducibly altered by BMI-1
knockdown in ESFT cell lines (Fig. 5B), only nidogen 1 expression
was altered atthe level of protein expression (Fig. 5C). Nidogen 1 is
acell adhesion protein and integral component of basement
membranes (38). To determine if nidogen 1 levels affect ESFT cell
adhesion, we examined the consequences of NID1 knockdown.
As shown in Fig. 5D, NID1 knockdown accelerated ESFTcell adhe-
sion, suggesting that repression of nidogen 1 may be functionally
important in BMI-1–mediated adhesion and tumorigenicity. More
extensive studies are now required to test this possibility.
Discussion
We have found that BMI-1 is highly expressed by ESFTcells and
that it promotes anchorage-independent growth and in vivo
tumorigenicity. Importantly, our studies reveal that these tumor-
igenic properties of BMI-1 are modulated independent of CDKN2A
repression, indicating that novel mechanisms of BMI-1 oncogenic
activity exist. In fact, in contrast to normal mesenchymal stem
cells, altering expression levels of BMI-1 has no significant or
consistent effect on CDKN2A or p16 expression in ESFT. Although
this may be an artifact of in vitro culture, it is also possible that
nonfunctional retinoblastoma family proteins prevent BMI-1–
mediated repression of CDKN2A in these cells (36). Although
previous reports have shown that pRB is only rarely mutated in
ESFT (25, 39), recent work suggests that pRB function may be
functionally inactivated by EWS-FLI1 itself (40). Further studies are
now required to determine if pRB inactivation contributes to the
dissociation of BMI-1 from p16 regulation in ESFT.
Although the histogenesis of ESFT remains a mystery, recent
studies implicate somatic stem cells as cells of origin (reviewed in
ref. 41). Given that most somatic stem cells express high-levels of
BMI-1 and that expression diminishes during differentiation (42), it
is possible that the high level of BMI-1 expression we observe in
ESFT cells is an inherent feature of their cellular origin.
Alternatively, expression of the EWS-FLI1 fusion oncogene may
be able, in some cell types, to induce BMI-1 as was recently shown
in NIH-3T3 cells (43). Cell type– and differentiation state–
appropriate experimental models are now required to test which
of these situations exists in the initiation of ESFT. EWS-FLI1–
mediated transformation of primary fibroblasts requires the
inactivation of p16-retinoblastoma and/or p53 pathways (44, 45).
We speculate that BMI-1–mediated repression of CDKN2A may
confer cellular tolerance to EWS-FLI1 in the ESFTcell of origin and
that this epigenetic inactivation of tumor suppressor pathways
could explain the relatively low incidence of secondary genetic
mutations in primary ESFT (3). In support of this, we find that
BMI-1 levels are, in general, higher among primary tumors
compared with ESFTcell lines (Fig. 1A), suggesting that mutations
in p16 and/or p53 that are more commonly present in cell lines
Figure 4. BMI-1 promotes ESFT tumorigenicity in vivo. A, NOD-SCID mice
injected with TC71 cells that express reduced levels of BMI-1 (shBMI1)show
delayedengraftmentandslowedinvivo growthcomparedwithmiceinjectedwith
control (shNS) transduced cells (two-tailed paired t test, P<0.001). B, time to
tumor cell engraftment is decreased and tumor growth enhanced in NOD-SCID
mice injected with TC71 cells that overexpress BMI-1 (pBp-Bmi1)compared
with empty vector (pBp-V) transduced cells (two-tailed paired t test, P=0.01).
C, QRT-PCR analysis of RNA harvested from tumor xenografts confirms
differential expression of BMI-1.
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131
(25, 46) may at least partially compensate for BMI-1 expression. It
has been previously documented that p16 loss in lung tumors
correlates with low BMI-1 expression (47). We are now testing
whether there is a relationship between BMI-1 expression and p16
and/or p53 status in primary ESFTand whether differences exist in
clinical presentation or outcome between tumors that express high
versus low levels of BMI-1.
We have shown that altering BMI-1 expression affects the ability
of both p16-null and p16-positive cells to form anchorage-
independent colonies in vitro and tumors in vivo.Incorroboration
with our findings, several recently published reports have revealed
that, in cooperation with other oncogenic lesions such as mutated
epidermal growth factor receptor or H-RAS, BMI-1 can transform
both CDKN2A wild-type and CDKN2A null cells (22, 23). In
addition, BMI-1 knockdown in p16-null DAOY medulloblastoma
cells significantly impedes tumor formation in vivo (19, 37). Thus,
although initial studies of BMI-1 implicated the repression of the
p16
Ink4a
/p14
ARF
-encoding CDKN2A locus as the primary mecha-
nism of oncogenic action (11, 12), more recent data from our lab
and others show a pivotal role for p16-independent mechanisms.
In order to identify potentially novel downstream targets of
BMI-1, we performed gene expression profiling of ESFT cells
following BMI-1 knockdown and compared BMI-1–responsive
genes to those genes similarly affected by BMI-1 knockdown in
human medulloblastoma cells (19). Although a significant subset of
genes was commonly regulated by BMI-1 in both tumor types,
otherswereuniquely alteredin only one of the model systems. This
implies that although some biological pathways are shared among
different tumor types, it is likely that at least some downstream
effectors of BMI-1 differ among tumors of different cellular origins.
Nevertheless, our findings show that significant commonalities
exist. In particular, direct comparison between ESFT and
medulloblastoma cells (19) reveals that alterations in cell adhesion
and extracellular remodeling processes are highly overrepresented
and common to both tumor types. For the current study, we have
validated that expression of the basement membrane protein
nidogen 1 is repressed by BMI-1 in ESFTcells. Nidogen 1 acts as a
linker between laminins, collagens, and proteoglycans in the
extracellular matrix and binds to cell surface integrins (38).
Interestingly, it has recently been reported that NID1 is frequently
silenced in colon cancer, suggesting that nidogen 1 may have a role
as a tumor suppressor gene, preventing invasion and metastasis
(48). In support of this possibility, we have found that down-
regulation of NID1 promotes adhesion of ESFT cells in vitro,
recapitulating the effects of BMI-1 overexpression. Therefore, we
hypothesize that the effect of BMI-1 knockdown on cell adhesion,
through modulation of nidogen 1 and/or other adhesion-related
proteins,is likelytounderliethedelaytoinvivo tumorengraftment
that we observe in ESFT cells with reduced levels of BMI-1.
Consistent with this hypothesis, delayed engraftment and altered
adhesion pathways have also been shown to be a feature of bmi-1–
deficient murine glioma cells (22). Thus, the cumulative evidence
suggests that modulation of adhesion molecules such as nidogen 1
is likely to contribute to the oncogenic function of BMI-1 in ESFT
as well as other tumor types.
Finally, polycomb genes, including BMI-1, play a central role in
the repression of differentiation and in the controlled orchestration
of normal development (5). Our finding that developmental
pathways are significantly affected by BMI-1 knockdown in ESFT
cells suggests that the embryonic function of BMI-1 is being
recapitulated in these undifferentiated tumor cells. It is particularly
noteworthy that both WNTand NOTCH pathway genes are highly
affected by BMI-1 knockdown as both of these developmental
pathways have been previously implicated in ESFT growth and
tumorigenicity (49, 50). Intriguingly, our data also show that among
the affected developmental processes, BMI-1 loss has its most
profound effect on genes that are involved in neural development
with BMI-1 knockdown leading to increased expression of neural
markers (Table 1; Supplementary Table S1). These findings
corroborate recent documentation of the effects of bmi-1 loss on
the phenotype and neural differentiation capacity of murine
gliomas (22). One of the many clinical mysteries surrounding ESFT
is the observation that they vary from highly undifferentiated
tumors to tumors with obvious neural features. It is tempting to
speculate that the phenotype of a particular tumor is either (a)
predetermined by the BMI-1 expression level in the parent cell
that originally acquires the EWS-ETS translocation or (b)a
consequence of the tumor microenvironment and its downstream
effects on BMI-1 expression. More extensive studies are required to
evaluate these hypotheses.
Table 1. Gene ontology biological processes significantly affected by BMI-1 knockdown in A4573 ESFT cells
No. Gene ontology biological process Douglas et al. (P)OverlapwithWiederschainetal.
1Development:neurogenesisingeneral 3.2E!07 *
2Proteolysis:connectivetissuedegradation 9.9E!05 *
3 Cell adhesion: cell-matrix interactions 1.0E!04 *
4Reproduction:progesteronesignaling 2.5E!04 NS
5Celladhesion:amyloidproteins 2.6E!04 *
6Signaltransduction:WNTsignaling 4.2E!04 NS
7Cytoskeleton:regulationofcytoskeletonrearrangement 5.5E!04 NS
8Proteolysis:ECMremodeling 2.3E!03 *
9Development:skeletalmuscledevelopment 2.3E!03 *
10 Cell adhesion: integrin-mediated cell-matrix adhesion 2.6E!03 NS
11 Signal transduction: neuropeptides signaling pathways 3.1E!03 NS
12 Signal transduction: NOTCH signaling 4.5E!03 *
Abbreviation: NS, nonsignificant overlap.
*Processes in which statistically significant overlap (P < 0.01) exists in BMI-1–modulated genes between ESFT and medulloblastoma cells (19).
BMI-1 Promotes Ewing Sarcoma Tumorigenicity
www.aacrjournals.org 6513 Cancer Res 2008; 68: (16). August 15, 2008
132
In summary, we have shown that BMI-1 functions as an
oncogene in ESFT and that it promotes tumorigenicity in a
CDKN2A-independent manner, influencing pathways involved in
cell adhesion, differentiation, and development. Given its central
role in the regulation of multiple developmental processes in
normal stem cells, we expect that no single gene will be uniquely
responsible for the oncogenic effects of BMI-1. Nevertheless, our
findings support the hypothesis that regulation of adhesion
pathways is central to BMI-1–mediated tumor promotion. Future
studies directed at understanding this relationship are likely to
proffer attractive and novel targets for therapeutic intervention
that may be common to the multiple human cancers that
deregulate and overexpress BMI-1.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Received 11/9/2007; revised 5/19/2008; accepted 6/11/2008.
Grant support:VFoundationandtheMargaretE.EarlyMedicalResearchTrust
(E.R. Lawlor), NIH grants U01 CA88199 and U01 CA115757 (T.J. Triche), California
Institute for Regenerative Medicine training grants CHLA T2-00005, USC T1-00004
(D. Douglas and J.H-R. Hsu), and NIH training grant T32 CA 09659 (J. van Doorninck).
Support from the My Brother Joey, Stop Cancer, and T.J. Martell Foundations is also
gratefully acknowledged.
The costsof publication of this article weredefrayed inpartbythe payment ofpage
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Figure 5. BMI-1 accelerates ESFT cell
adhesion in vitro. A, A4573 cells were
plated in 96-well plates and monitored for
adhesion. Cells with stable knockdown of
BMI-1 (shBMI1)showreducedadhesion
relative to control cells (shNS; top),
whereas adhesion is accelerated in
BMI-1–overexpressing cells (pBp-BMI1) in
comparison with empty vector cells (pBp-V;
bottom). Points, mean absorbance of
crystalviolettakenupbyadherentcellsinat
least seven replicate wells from duplicate
experiments at 30-min intervals.
B, QRT-PCR validates that two cell
adhesion genes identified by microarray
analysis are truly BMI-1–responsive.
Note that although basal levels of the two
genes differ, knockdown of BMI-1 leads to
significant up-regulation of NID1 and
down-regulation of VEZT in all three ESFT
cell lines. C, Western blot confirms the
up-regulation of nidogen 1 protein in A4573
cells following BMI-1 knockdown (siBMI1A
and siBMI1B) and down-regulation in
the presence of BMI-1 overexpression
(pBp-BMI1). D, A4573 cells were
transfected with siNID1 or siNS control
oligonucleotides and knockdown confirmed
after 48 h by QRT-PCR (left). Transfected
cells were plated in 96-well plates and
adhesion measured as described in A.
Reduced expression of NID1 accelerates
adhesion.
Cancer Research
Cancer Res 2008; 68: (16). August 15, 2008 6514 www.aacrjournals.org
133
The authors thank Heinrich Kovar, Darwin Prockop, Carolyn Lutzko, Gerard Evan,
andRobertIlariaforcellsandreagents.WealsothankmembersoftheTriche&Lawlor
labs for helpful discussion, and Betty Schaub and members of the genomics, animal,
FACS, and vector cores for technical assistance. Tumor samples were provided by the
CHLADepartmentofPathologyandbytheChildren’sOncologyGroupBiorepositoryin
Columbus, OH. We gratefully acknowledge the staff of these facilities for their efforts.
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BMI-1 Promotes Ewing Sarcoma Tumorigenicity
www.aacrjournals.org 6515 Cancer Res 2008; 68: (16). August 15, 2008
Abstract (if available)
Abstract
The stem cell self-renewal pathway is often hijacked and over-activated in cancer cells. The polycomb group protein BMI-1 is a known regulator of stemness and its over-expression is associated with a number of malignancies. In this study, we have examined the role of BMI-1 in the pathogenesis of Ewing's sarcoma family of tumors (ESFT). We hypothesize that deregulation of BMI-1 -driven signaling pathway is central to the initiation and maintenance of this family group of aggressive pediatric tumor. To assess our hypothesis, we have performed gain and loss of function studies and provided evidence to support the role of BMI-1 in promoting anchorage-independent growth of ESFT in vitro and tumorigenicity in vivo independent of the genetic status of the cells. Contrary to early indications in stem cells and other caner cell types, BMI-1 promotes growth of ESFT cells independent of CDKN2A repression. To identify novel BMI-1 downstream targets involved in BMI-1 mediated tumorigenicity, we have conducted gene expression profiling on ESFT cells following BMI-1 knockdown. Significantly, we discover hundreds of putative BMI-1 targets including genes involved in cell adhesion, development and differentiation. In particular, genes associated with cell-cell and cell-matrix adhesion are over-represented. In light of this data, we have focused our investigation on the role of BMI-1 in cell adhesion. Using in vitro adhesion assays, we find that BMI-1 accelerates ESFT adhesion in part through repression of the basement protein Nidogen 1 and a member of the WNT protein family, WNT5A. These studies indicate that BMI-1 may promote tumor growth and engraftment through modulation of adhesion pathways and non-canonical WNT signaling.
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Asset Metadata
Creator
Hsu, Hao-Ru Jessie
(author)
Core Title
Harnessing the power of stem cell self-renewal pathways in cancer: dissecting the role of BMI-1 in Ewing’s sarcoma initiation and maintenance
School
Keck School of Medicine
Degree
Doctor of Philosophy
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Genetic, Molecular and Cellular Biology
Publication Date
06/08/2010
Defense Date
05/19/2010
Publisher
University of Southern California
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Tag
BMI-1,contact inhibition,Ewing sarcoma,EWS-FLI1,OAI-PMH Harvest,polycomb group protein, CDKN2A, p16,stem cell pathways,YAP,Yes-associated protein
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Johnson, Deborah L. (
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), Frenkel, Baruch (
committee member
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committee member
), Stallcup, Michael R. (
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haoruhsu@usc.edu,jessiehhsu@gmail.com
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Tags
BMI-1
contact inhibition
Ewing sarcoma
EWS-FLI1
polycomb group protein, CDKN2A, p16
stem cell pathways
YAP
Yes-associated protein