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Investigating the roles of BMI-1 and IGF-1 in promoting ESFT survival
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Investigating the roles of BMI-1 and IGF-1 in promoting ESFT survival
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Content
INVESTIGATING THE ROLES OF BMI-1
AND IGF-1 IN PROMOTING ESFT SURVIVAL
by
Darren Jason Russell
___________________________________________________________
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(EXPERIMENTAL AND MOLECULAR PATHOLOGY)
May 2011
Copyright 2011 Darren Jason Russell
ii
ii
Table of Contents
List of Figures iii
Abstract iv
Introduction 1
Ewing’s Sarcoma 1
IGF-1 Signaling 1
BMI-1 and Cancer Stem Cells 4
BMI-1 Expression is Negatively Correlated with IGF-1 Axis 7
Signaling
My Work 9
Chapter 1: Materials and Methods 10
Chapter 2: Results 12
BMI-1 Cell Lines are More Sensitive to IGF-1R Inhibition 12
Effect of IGF-1R Blockade on ESFT Viability 14
Mechanism of Decreased Viability 18
BMI-1 and IGF-1 Cooperate to Promote Akt Phosphorylation 22
Conclusion 24
References 30
Bibliography 34
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iii
List of Figures
Figure 1: Overview of IGF-1R/PI3K/Akt signaling 3
Figure 2: Overview of BMI-1 6
Figures 3A-3B: Gene expression profiling studies demonstrate distinct 9
molecular signatures of BMI-1-low tumors
Figures 4A-4C: BMI-1 positive cell lines are sensitive to IGF-1R blockade 13
Figures 5A-5C: BMI-1 knockdown does not decrease viability 16
Figures 6A & 6B: BMI-1 knockdown confers sensitivity to IGF-1R blockade 17
Figure 7A & 7B: IGF-1R inhibition causes apoptosis in BMI-1 knockdown cells 19
Figure 8: BMI-1 and IGF-1 cooperate to promote Akt phosphorylation 22
Figures 9A-9D: Proposed model of interaction between BMI-1 and 29
IGF-1 signaling
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Abstract
Purpose: Ewing sarcoma family tumors (ESFT) are highly aggressive pediatric and
young adult stem cell tumors for which prognostic biomarkers and novel treatments are
needed. New treatments targeting the IGF-1R signaling pathway show promise, but may
not be effective in treating all patients. We have assessed the potential clinical
significance of BMI-1 expression level on treatments utilizing IGF-1R inhibition.
Experimental Design: BMI-1-positive and –negative ESFT cell lines were identified
using PCR, Q-RT-PCR, and western blot analysis. Sensitivity to IGF1-R inhibition was
determined using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium] assays. Performing gain and loss of function studies
assessed the role of BMI-1 on IGF-1R inhibition. The effect of IGF-1 inhibition on cell
viability was evaluated by PARP cleavage western blots. Cooperation between BMI-1
and IGF-1 was assessed by performing immunoblotting to detect Akt phosphorylation.
Results: BMI-1 expression is negatively correlated with IGF-1R axis signaling in ESFT.
Significantly, although BMI-1-positive cell lines are more sensitive to IGF-1R inhibition
than BMI-1-negative cells, overexpressing BMI-1 decreased this sensitivity, while
knocking down BMI-1 expression increased the effects of IGF-1R inhibition. PARP
cleavage western blots showed that the likely mechanism of decreased viability in cell
lines treated with IGF-1R inhibition is apoptosis. Consistent with these data, we have
shown that BMI-1 and IGF-1 cooperate to promote Akt phosphorylation.
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Conclusion: BMI-1 and IGF-1 signaling cooperate to promote ESFT survival and
tumorigenesis through activation of the PI3K/Akt survival pathway.
1
1
Introduction
Ewing’s Sarcoma
Ewing’s sarcoma family tumors (ESFT) are highly aggressive pediatric and young
adult tumors. It is the second most common bone tumor of childhood and adolescence,
with an incidence of 2.93 cases per 1,000,000 in the US [1-2]. ESFT can arise in any
bone or soft tissue, most commonly the long, flat bones, and 25% of patients present with
metastasis at diagnosis [3-4]. While conventional cytotoxic chemotherapy has improved
survival form 10% to 75% in patients with localized disease, this treatment modality still
fails in 25% of patients with localized tumors and an overwhelming 75% of patients with
metastatic disease [3, 5]. ESFT is characterized by chromosomal translocations, most
commonly EWS-Fli1 (90-95%), and EWS-ERG (5-10%), that give rise to EWS/ETS
chimeric fusion proteins that disrupt normal tissue development acting as abnormal
transcription factors, repressors, or altering the processing of RNA [6]. Among the
activities of EWS/Fli1 is the up-regulation of insulin-like growth factor-1 (IGF-1),
driving ESFT cell proliferation and survival [6].
IGF-1 Signaling
The IGF-1 signaling pathway is an important process in normal cell growth and
differentiation, but also plays a role in cancer biology driving transformation and anti-
apoptotic signaling. While expression of IGF-1 is expressed by a wide variety of tumor
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cell types, ESFT also expresses high levels of the insulin-like growth factor receptor
(IGF-1R), creating autocrine stimulatory feedback loops of IGF-1 signaling.
The IGF-1R is a transmembrane homotetramer consisting of two extracellular α
subunits and two transmembrane β subunits. IGF-1R binds to its ligand, IGF-1R with
high affinity, resulting in initiation of the IGF-1 signaling cascade. Upon binding, the
IGF-1R tyrosine kinase domain becomes phosphorylated, which acts to phosphorylate the
intracellular proteins SHC and IRS 1-4, leading to activation of MAPK and PI3K/Akt
pathways, respectively [50].
IGF signaling is tightly controlled by a series of regulatory proteins including two
receptors, two ligands, at least six IGF-binding proteins (IGFBPs), and IGFBP-related
proteins including proteases. Only IGF-1R activates IGF signaling, as IGF-2R acts to
control soluble ligand levels by targeting IGF-2 for lysosomal destruction. Ligand
bioavailability is further controlled by IGFBPs, namely IGFBP3, which bind to and
protect IGFs from proteolytic degradation. Prior to binging their receptors IGFs are freed
from IGFBPs by specific proteases.
IGF-1 signaling is further amplified by the action of EWS-Fli1 binding to and
repressing the IGF binding protein 3 (IGFBP3) promoter, as IGFBPs control IGF-1
bioavailability[3].
Upon ligand binding and activation, phosphorylated IRS becomes a docking site
for the p85 and p110 subunits of PI3K leading to conversion of phosphatidylinositol-4,5-
bisphosphate (PIP2) to phosphatidylinositol-3,4,5-biphosphate (PIP3) (Fig. 1). PIP3
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3
Figure 1. Overview of IGF-1R/PI3K/Akt signaling.
Following ligand binding, IGF-1R stimulates the PI3K/Akt signaling pathway leading to
cell survival, proliferation, metabolism, protein synthesis, and cell growth.
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activates phosphoinositide-dependent kinase-1 (PDK1), which acts to activate Akt
through phosphorylation of specific serine and threonine residues. Once activated,
phospho-Akt inhibits BAD, GSK3, and p27, thus promoting survival, metabolism, and
proliferation, respectively.
Alternately, phosphorylation of SHC by activated IGF-1R, along with Grb2 and
SOS activate Ras. Ras activates Raf, which phosphorylates and activates MAPK/ERK
kinases (MEK1-2), which phosphorylate and activate extracellular signal-related kinase-1
and -2 (ERK1-2), leading to activation of proteins that promote a more aggressive
phenotype in tumors.
BMI-1 and Cancer Stem Cells
In addition to the activities of the EWS-Fli1, development of ESFT involves
inappropriate activation of stem cell genes that are not normally activated in mature
tissues. Like their stem cell counterparts, cancer stem cells expressing these genes
provide a means of initiation and maintenance of tumors, which thrive due to divisions of
these cancer stem cells [1, 8-9]. These cancer stem cells hijack stem cell self-renewal
pathways resulting in development of neoplasms, tumor progression, metastasis, and
tumor recurrence [10-13].
Among the stem cell genes expressed by ESFT is the polycomb ring finger
oncogene BMI-1. As part of the polycomb repressive complex 1 (PRC1), BMI-1 acts to
maintain stable repression of genes through epigenetic chromatin modification [8, 14-15].
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In addition, BMI-1 promotes anchorage-independent growth and tumorigenicity in ESFT,
and alters developmental and cell adhesion pathways [17].
BMI-1 has emerged as a key player in cancer stem cell biology and has been
shown to be highly expressed in pediatric brain tumors [18-19] and neuroblastoma [20],
and is required for proliferation of cancer stem cells in leukemia [12] and nasopharyngeal
carcinoma [21].
BMI-1 was first identified in B-cell lymphomas where it cooperates with c-myc in
cancer initiation [30-31]. The three dimensional structure of BMI-1 consists of a
conserved N-terminal RING finger domain and a central helix-turn-helix-turn-helix-turn,
which allows it to interact with a DNA telomerase, leading to cell immortalization [33-
36]. Notably, BMI-1 acts as an anti-apoptotic mediator in cancer [32], and plays a role in
self-renewal and differentiation in human hematopoietic and LSCs [37-41].
Although the molecular mechanisms used by BMI-1 to promote cancer stem cells
is not fully elucidated, it has been shown to repress the Cdkn2a locus and downregulate
expression of p16INK4a and p19ARF [42-45] (Fig. 2). Downregulation of p16INK4a
and p14ARF allow cancer cells expressing BMI-1 to progress unchecked through the cell
cycle free of internal or external anti-proliferative stimuli.
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6
Figure 2. Overview of BMI-1.
Transcriptional repression of p16INK4a and p14Arf by BMI-1 results in escape from cell
cycle arrest and apoptosis by overriding the Rb and p53 cell cycle checkpoints,
respectively.
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P16INK4a acts as a cell cycle inhibitor by binding and inactivating Cdk4 and
Cdk6. This inhibitory effect suppresses phosphorylation of Rb, arresting the cell cycle in
G1/S phase [46-47]. P14ARF also inhibits the cell cycle by stabilizing p53, leading to
cell cycle arrest in G1 and G2/M and apoptosis [48-49]. In repressing the transcription of
p16INK4a and p14ARF, BMI-1 overrides the p16/Rb and p14/p53 tumor suppressor
pathways and promotes cell cycle progression and proliferation.
While suppression of the p16/Rb and p14/p53 pathways may be sufficient to
bypass cell cycle checkpoints, our lab has recently reported that BMI-1 is over-expressed
by the majority of ESFT and acts to promote tumorigenicity in a p16INK4a/p14ARF-
independent manner [17, 22].
BMI-1 Expression is Negatively Correlated with IGF-1 Axis Signaling
In complementary work in the lab it was discovered that 80% of primary ESFT
tumors over-express BMI-1. In contrast, in a minority of tumors BMI-1 is either not
expressed or expressed to only very low levels (Fig. 3A). Global gene expression
microarrays were conducted in order to compare global gene expression between ESFT
expressing high and low/negative levels of BMI-1 (Fig. 3B). Over 4000 genes were
identified as being differentially expressed between BMI-1-positive and BMI-1-negative
tumors. In order to refine the list to identify genes and pathways that are most likely to be
of biologic and functional relevance, gene specific enrichment analysis was performed.
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Figures 3A-3C: Gene expression profiling studies demonstrate distinct molecular
signatures of BMI-1-low tumors.
A. BMI-1 raw signal intensities from 5 BMI-1-low and 5 BMI-1-high primary tumors
(arbitrary units). B. Principal components analysis of 10 tumors using all core
transcripts segregates BMI-1-low and BMI-1-high tumors into two distinct clusters.
Cluster ellipses encompass 2 standard deviations. C. BMI-1-low tumors display down-
regulation of genes in the IGF1 pathway relative to BMI-1-high tumors. The fold change
in median signal intensity for each gene was calculated for BMI-1-high and BMI-1-low
tumors relative to median expression in all 10 tumors. Error bars represent SEM for 5
tumors. Source: Cooper et al.
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Importantly, results of this analysis demonstrated that genes in the IGF-1 signaling
pathway were highly significantly (p<0.001) over-represented among differentially
expressed genes. Specifically, expression profiling revealed downregulation of genes
involved in IGF-1 signaling among primary tumors and cell lines with a BMI-1-negative
phenotype (Fig. 3C) [22].
My Work
In this study, we have investigated the signaling pathways associated with the
presence of BMI-1 expression. We have identified a correlation between BMI-1
expression levels and IGF-1 axis signaling and have identified a unique molecular
subclass of ESFT. Further, we have observed a possible mechanistic relationship
between BMI-1 and IGF-1 signaling which promotes survival in ESFT. In particular
these data support our hypothesis that BMI-1 and IGF-1 cooperate to promote ESFT
survival through activation of Akt.
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Chapter 1: Materials and Methods
Cell culture
TC71, CHLA9, and TC248 cell lines were provided by Dr. Timothy Triche. Cells
were cultured in monolayers on tissue culture-treated plates in RPMI 1640 supplemented
with 10% FBS, L-glutamine, and pen/strep. Cells were grown to confluence and passaged
using 0.5% trypsin in EDTA.
Quantitative real-time reverse transcription PCR
Quantitative real-time reverse transcription PCR was performed by Dr. Jessie
Hao-Ru Hsu, as described [17]. In brief, cDNA generated from DNase I-treated RNA
was analyzed in triplicate using quantitative real-time reverse transcription PCR (QRT-
PCR) using TaqMan Gene Expression Assays (Applied Biosystems).
Viability assays
Cells growing in log phase were plated onto 96 well plates at a concentration of 5
X 103 cells per well and allowed to attach overnight. Cells were plated in 100µl RPMI
1640 supplemented with 5% FBS, pen/strep, and L-glutamine. Attached cells were
treated for a period of 48 hours with increasing concentrations of picropodophillin (PPP;
Enzo Life Sciences), IMC-A12 (ImClone) or DMSO vehicle dissolved in 100 µl of
media. Viability was analyzed using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays (CellTiter 96 Aqueous
One Solution Cell Proliferation Assay; Promega) according to manufacturer protocol.
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Viability assays were performed at least five times with a minimum of six wells counted
per condition.
Gene knockdown and over-expression
Knockdown and over-expression of BMI-1 in TC71 cells were performed by
Long Hung and Dr. Jessie Hao-Ru Hsu, as described [17]. In brief, TC71 cells were
transfected with BMI-1-targeted small interfering RNA (siRNA) or negative control
siRNA (Ambion). Stable knockdown was achieved by siRNA sequences cloned into
pSuper-retro-puro short-harpin vector backbone. For BMI-1 overexpression, TC71 BMI-
1 cDNA was amplified by PCR and cloned into pBabe-Puro. TC71 BMI-1 knockdown
and over-expression cells were cultured in media containing 2 µg/mL of puromycin.
Protein lysate isolation
Whole cell lysates were collected from 10 cm plates using a cell lifter and washed
with 10 ml cold PBS. Cells were lysed using RIPA buffer supplemented with a protease
inhibitor phosphatase inhibitor cocktail and 1% sodium orthovanadate in HBSS.
Immunoblotting
Western blots were performed using whole cell lysates. Primary antibodies used
were as follows: Anti-Bmi-1, clone F6 (Millipore); PARP, Phospho-Akt (Ser473), and
GAPDH (Cell Signaling). All antibodies were diluted 1:1000 in 5% milk or 1% BSA in
TBST (Phospho-Akt).
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Chapter 2: Results
BMI-1 Positive Cell Lines are More Sensitive to IGF-1R Inhibition
The observation of downregulation of the IGF-1 signaling axis in BMI-1-negative
ESFT samples led us to hypothesize that inhibition of the IGF-1 receptor (IGF-1R) would
be relatively ineffective in suppressing the growth of ESFT cells that express low levels
of BMI-1.
To test this hypothesis, I examined the viability of cell lines representative of
BMI-1-positive and BMI-1-negative tumors treated with the small molecule IGF-1R
inhibitor picropodophyllin (PPP) and the anti-IGF1-R antibody IMC-A12. Quantitative
reverse transcription polymerase chain reaction (Q-RT-PCR) of ESFT cell lines revealed
that TC71 and CHLA9 express high levels of BMI-1 and that TC248 expresses low BMI-
1 levels, making them suitable models of BMI-1-positive and negative primary ESFT
tumors, respectively (Figs. 4A & 4B). TC71, CHLA9, and TC248 were treated with the
IGF-1R small molecule inhibitor PPP, DMSO as a vehicle control, or the anti-IGF-1R
monoclonal antibody IMC-A12 for 48 hours and their viability was assayed by MTS
assay. MTS viability assay was chosen because it allows the relative viability of cell
populations to be quantified by means of converting a tetrazolium compound ([3-(4,5-
dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner
salt; MTS]) to colorimetric media-soluble formazan in metabolically active cells. This
assay was also particularly attractive because it is suitable for non-adherent or partially
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Figure 4A-4C. BMI-1 positive cell lines are sensitive to IGF-1R blockade.
A. RT-PCR of ESFT cell lines reveals differential expression of BMI-1. TC71 and
CHLA9 are BMI-1-positive, TC248 is BMI-1-negative. Expression levels were assayed
in triplicate and normalized relative to GAPDH isolated from the same samples.
Columns, averages of triplicates; bars, SD. B. QRT-PCR of ESFT cell lines reveals
differential expression of BMI-1. TC71 and CHLA9 are BMI-1-positive, TC248 is BMI-
1-negative. C. PPP and IMC-A12 significantly decrease viability of both TC71 and
CHLA9 but not TC248 cells. Cells were treated with PPP or IMC-A12 at increasing
concentrations for 48 hours and viable cells relative to controls were plotted. BMI-1-high
TC71 and CHLA9 cells are compared at each concentration to BMI-1-low TC248 cells.
Points, average viability of six replicate experiments; bars, SEM.
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adherent cells, as treatment with IGF-1R inhibitors caused many of the cells to become
detached from tissue culture plates. These experiments revealed that BMI-1-negative
cells were in fact more resistant to IGF-1R blockade by both PPP and IMC-A12 (Fig.
4C). When treated with PPP, TC79 and CHLA9 had an IC50 of 500 nM and 1 uM,
respectively, while the BMI-1-negative TC248 cell line did not reach 50% growth
inhibition at even the highest concentration of the drug. Similarly, The BMI-1-positive
TC71 and CHLA9 cell lines were affected more than TC248 when treated with IMC-
A12, with TC71 reaching 50% viability at the lowest dose (25 nM), while TC248 was
virtually unaffected by the anti-IGF-1R antibody. Further, ESFT cell lines sensitivity to
IGF-1R blockade was dependent on BMI-1 expression levels, with higher expression of
BMI-1 correlating with increased sensitivity to IGF-1R blockade by both PPP and IMC-
A12. Although this observation does not prove causality, it does support a relationship in
which BMI-1 expression affects the IGF-1 signaling pathway.
Thus, these findings show that ESFT cells that express low levels of BMI-1 are
less sensitive to IGF-1R blockade, and that levels of BMI-1 expression correlate with the
observed response to this treatment modality.
Effect of BMI-1 and IGF-1R Blockade on ESFT Viability
The apparent BMI-1 dose-dependent response of ESFT cell lines to PPP and
IMC-A12 led to the hypothesis that BMI-1 is responsible for conferring sensitivity to
IGF-1R inhibition. If this hypothesis is true then loss of BMI-1 in BMI-1-positive cells
should result in reduced sensitivity to the growth inhibitory effects of IGF-1R blockade.
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To test this hypothesis I performed gain and loss of function studies. TC71 cells
expressing high levels of BMI-1 were treated with BMI-1 targeted RNAi, and BMI-1
over-expression vectors to knock down and over-express BMI-1, respectively. First,
BMI-1 knockdown and over-expression were confirmed by analyzing BMI-1 protein
levels by immunoblotting using anti-BMI-1 antibodies (Fig. 5A,B). MTS cell viability
assays showed no difference between control and BMI-1 knockdown cells (Fig. 5C)
confirming that loss of BMI-1 does not, by itself, inhibit TC71 cell growth. Next, to test
the role of BMI-1 in conferring sensitivity to treatments targeting the IGF-1R signaling
axis, the viability of TC71 cells with BMI-1 knockdown and over-expression treated with
PPP, IMC-A12, and DMSO vehicle control was analyzed by MTS cell viability assay.
Contrary to my hypothesis that BMI-1 confers sensitivity to IGF-1R blockade, I found
that TC71 cells expressing the BMI-1 silencing RNAi showed lower viable cell number
after treatment with PPP and IMC-A12 than non-silencing control cells (Fig. 6A).
Further, TC71 cells over-expressing BMI-1 were more resistant than their wildtype
counterparts when treated with the small molecule tyrosine kinase inhibitor PPP, as BMI-
1 knockdown cells had approximately 23% less viable cells at 100 nM PPP than non-
silencing control cells treated with the same concentration of PPP. However this
protective effect is not seen in cells treated with the monoclonal antibody IMC-A12 (Fig.
6B). Taken together, these assays support a model in which high levels of BMI-1 are
associated with resistance to IGF-1R blockade in a BMI-1 dose-dependent fashion. As
such, knockdown of BMI-1 in these BMI-1-positive cells is associated with increased
rather than decreased sensitivity to IGF-1R inhibition.
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Figures 5A-5C. BMI-1 knockdown does not decrease viability.
A. Western blot analysis confirms siRNA-mediated knockdown of BMI-1 in TC71 cells
(sh BMI-1) relative to cells treated with non-silencing control siRNA (sh NS). Bar graph
represents densitometry of two replicates. Columns, average BMI-1 expression levels of
duplicate experiments relative to GAPDH; bars, SEM. B. Western blot analysis confirms
increased expression of BMI-1 in TC71 cells treated with overexpression vector (pBp
BMI-1) relative to cells treated with empty vector (pBp V). Columns, average BMI-1
expression levels of duplicate experiments relative to GAPDH; bars, SEM. C. siRNA-
mediated knockdown of BMI-1 in TC71 cells does not affect cell proliferation or death.
Bars, average of six replicates; bars, SEM.
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Figure 6A & 6B. BMI-1 knockdown confers sensitivity to IGF-1R blockade.
A. siRNA mediated knockdown of BMI-1 cells increases their sensitivity to both PPP and
IMC-A12 treatment relative to non-silencing control cells. TC71 sh BMI-1 and control
cells were treated for 48 hours with increasing concentrations of PPP or IMC-A12 and
viable cell number assessed using MTS assays. Points, average of six replicate
experiments; bars, SEM. B. Overexpression of BMI-1 decreases sensitivity of TC71
cells to PPP, but not to IMC-A12. TC71 pBp BMI-1 and control cells were treated for 48
hours with increasing concentrations of PPP or IMC-A12 and viable cell number assessed
using MTS assays. Points, average of six replicate experiments; bars, SEM.
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These findings are in conflict with our studies of BMI-1 negative (TC248) cells and
suggest that the reduced sensitivity to IGF-1R inhibition in TC248 cells is likely to be
due to mechanisms other than BMI-1 expression. In fact, is noteworthy that multiple
pathways were found to be differentially activated in BMI-1-negative tumors compared
to BMI-1-positive tumors. Therefore, we hypothesize that there are fundamental
differences in the biology of BMI-1-negative tumors that require their designation as a
unique molecular subclass. Other studies in the lab continue to address this hypothesis.
Because the vast majority of ESFT primary tumors and cell lines are BMI-1 positive, I
elected to use the BMI-1-positive cell line TC71 for the remainder of my experiments.
Mechanism of Decreased Viability
The MTS cell viability assays performed to examine the affects of anti-IGF-1R
treatments allow for the quantification of viable cell number among samples by
examining the mitochondrial metabolic activity of cells. This assay does not, however,
reveal the mechanism of the increase or decrease in viable cell number that was observed.
Decreases in viable cell number detected by MTS assays may be from a variety of
mechanisms such as senescence, autophagy, apoptosis, necrosis, decreased proliferation,
or a combination of these. In order to determine which mechanisms are responsible for
the decreased viability of cells treated with IGF-1R blockade it was necessary to perform
additional assays.
Because I observed membrane blebbing when I microscopically examined cells
treated with PPP and IMC-A12 (Fig. 7A), I hypothesized that the mechanism affecting
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Figures 7A & 7B. IGF-1R blockade causes apoptosis in BMI-1 knockdown cells.
A. phase contrast images of TC71 sh BMI-1 cells treated for 48 hours with 250 nM PPP
or DMSO vehicle. Treatment with PPP causes TC71 sh BMI-1 cells to bleb, whereas
DMSO treated control cells appear healthy. B. western blot analysis confirms significant
PARP cleavage in BMI-1 knockdown cells (sh BMI-1) treated with PPP. Cells
overexpressing BMI-1 (pBp BMI-1) and control cells (sh NS, pBp V) display little to
undetectable PARP cleavage. Bar graph represents densitometry analysis of western
blot. Bars, percent of PARP present in cleaved form relative to total PARP.
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the viability of these cells was apoptosis, as membrane blebbing is one of the hallmark
characteristics of cells undergoing apoptosis. There are several assays that can confirm
or exclude apoptosis in cells, including assays to detect trypan blue exclusion, PARP
cleavage, caspase cleavage, annexin V binding, and TUNEL staining. In order to confirm
apoptosis as the mechanism of decreased viable cell number in cells treated with IGF-1R
inhibition I performed a PARP cleavage western blot on BMI-1 knockdown cells and
non-silencing control cells treated with PPP or a DMSO vehicle only control (Fig. 7B).
PARP is a 116 kDa nuclear protein that is cleaved during apoptosis, yielding an 89 kDa
cleavage product. Apoptotic PARP cleavage can be detected by exposing cell lysates to
antibodies targeted against PARP. When uncleaved, PARP appears as a high molecular
weight band, while after cleavage PARP appears as a lower weight band indicating its
lower molecular weight cleavage product. Although PARP cleavage by itself is not
sufficient proof for apoptosis, the previously stated observation of membrane blebbing
suggests apoptosis as a likely mechanism of decreased viable cell number.
PARP cleavage analysis of TC71 cells revealed significant PARP cleavage in
cells with BMI-1 knockdown when treated with PPP. Further, little or no PARP cleavage
was detected in either non-silencing control cells, TC71 cells over-expressing BMI-1, or
TC71 cells transfected with an empty vector. The finding that levels of cleaved PARP
were significantly higher in BMI-1 knockdown cells treated with IGF-1R inhibition than
in either untreated BMI-1 knockdown cells or PPP treated non-silencing control cells is a
critical observation in this assay. This result suggests cooperation between BMI-1 and
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IGF-1 signaling, as the cytotoxic effect of PPP is amplified by the knockdown of BMI-1
in TC71 cells.
Together, these results support my hypothesis that knockdown of BMI-1 in TC71
cells increases sensitivity to treatment with IGF-1R blockade and that the mechanism of
decreased viability in these cells is likely due to apoptosis. Although membrane
exclusion and PARP cleavage assays suggest that apoptosis is the mechanism of
decreased viability, it would be useful to perform other means of detecting apoptosis such
as annexin V staining in order to rule out other means of cell death and BrdU assays to
determine whether there is also an effect on proliferation, as it is possible that more than
one mechanism of decreased viability is responsible.
BMI-1 and IGF-1 Cooperate to Promote Akt Phosphorylation
Because signaling through IGF-1R is an upstream regulator of Akt
phosphorylation, I hypothesized that the cooperation of BMI-1 and IGF-1R signaling
converges on phosphorylation of Akt. Previous publications have shown that
upregulation of BMI-1 activates the PI3K/Akt/GSK-3β pathway in nasopharyngeal
epithelial cells [21], suggesting activation of Akt as a likely point of cooperation between
BMI-1 and IGF-1 signaling. Akt phosphorylation has been shown to drive survival, cell
cycle progression, metabolism and angiogenesis. To determine if Akt is the point of
BMI-1/IGF-1 interaction we treated BMI-1 knockdown cells and non-silencing control
cells with PPP or a DMSO vehicle only control and examined the phosphorylation of Akt
by immunoblotting (Fig. 8).
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Figure 8. BMI-1 and IGF-1 cooperate to promote Akt phosphorylation.
Western blot analysis confirms that siRNA-mediated knockdown of BMI-1 (sh BMI-1) in
cells treated with PPP results in lower levels of Akt phosphorylation than in cells treated
with BMI-1 knockdown alone or control cells (sh NS). Bar graph represents densitometry
of western blots. Columns, average levels of pAkt relative to GAPDH isolated from the
same cells.
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Immunoblotting assays to detect Akt phosphorylation showed that BMI-1 knockdown
cells treated with PPP display lower levels of Akt phosphorylation than untreated cells.
The BMI-1 knockdown cells also displayed lower Akt phosphorylation than non-
silencing control cells treated both with PPP and with a DMSO vehicle only control. The
finding that knocking down BMI-1 in TC71 cells and subjecting them to IGF-1R
inhibition resulted in decreased phosphorylation of Akt, helps to support my hypothesis
that the Akt pathway is likely to be the point at which BMI-1 and IGF-1 signaling
cooperate to drive ESFT cell survival.
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Conclusion
Ewing’s sarcoma is a highly aggressive tumor and although the majority of
patients with localized disease respond favorably to conventional therapy consisting of
cytotoxic chemotherapy and radiation therapy, patients with metastatic disease or
relapsed disease fair poorly with this treatment modality[3]. The observation that ESFT
upregulates IGF-1 and IGF-1R, and the extensive knowledge of downstream pathways
have made inhibitors of IGF-1R an ideal target for molecular therapy. With well over 30
anti-IGF-1R therapies in pre-clinical and clinical trials, treatments targeting IGF-1R are
emerging as promising new tools in the fight against Ewing’s sarcoma and other
tumors[23]. Clinical trials using small anti-IGF-1R antibodies have revealed the caveats
that these treatments may not benefit all patients to the same degree and that patients
likely to benefit need to be identified using predictive biomarkers[7, 23-26].
In this our lab we have shown that the level of BMI-1 expression in ESFT
primary tumors and cell lines inversely correlates with expression of genes involved in
the IGF-1 signaling pathway. While the majority of ESFT express high levels of BMI-1,
there exists a subset of BMI-1-negative or BMI-1-negative tumors and cell lines that
display significantly different gene expression profiles and differential expression of
genes involved in important biological pathways[22]. The down-regulation of genes
involved in IGF-1 signaling identifies BMI-1 as a potential predicative biomarker for
IGF-1R inhibitor response. Importantly, ESFT primary tumors and cell lines expressing
little or no BMI-1 may represent a patient population of non-responders requiring a new
molecular sub-classification based on BMI-1 expression. Our finding that the BMI-1
25
25
negative TC248 cell line is relatively insensitive to both PPP and IMC-A12 warrants
further investigation into the biological differences between BMI-1-positive and BMI-1-
negative ESFT.
Because BMI-1 is robustly over-expressed in 80% of ESFT, we focused the
remainder of our studies on the BMI-1-positive cell line TC71. BMI-1 knockdown and
over-expression TC71 cells allowed us to study the role of BMI-1 in ESFT cell lines.
Our data suggests that BMI-1 is mechanistically responsible for conferring
resistance to IGF-1R inhibition. TC71 cells with BMI-1 knockdown showed increased
sensitivity to IGF-1R blockade by both the small molecule tyrosine kinase inhibitor PPP
and the anti-IGF-1R antibody IMC-A12. Further, TC71 cells over-expressing BMI-1
showed augmented resistance suggesting that BMI-1 is mechanistically responsible for
the observed drug resistance. To further examine this phenomenon, additional similar
experiments with additional cell lines are needed.
The small molecule inhibitor PPP and the anti-IGF-1R monoclonal antibody
IMC-A12 produce their cytotoxic effects on malignant cells through different
mechanisms. PPP inhibits the autophosphorylation of the IGF-1R, likely at the substrate
level by mimicking IGF-1R tyrosines and inhibiting their phosphorylation[27]. In this
way PPP inhibits IGF-1R phosphorylation and downstream signaling. In addition to
inhibiting tyrosine phosphorylation PPP degrades the IGF-1R [28]. In contrast, IMC-
A12 is an anti-IGF-1R antibody that exerts it effect by binding the IGF-1R and blocking
ligand binding. In doing so IMC-A12 blocks PI3K/Akt signaling resulting from IGF-1
stimulation. IMC-A12 also causes IGF-1R internalization, thus reducing the number of
26
26
available receptors of its ligand[7]. Because ESFT relies on activation of the PI3K/Akt
pathway for cell proliferation and protection from apoptosis [3], inhibiting IGF-1R
reduces the anti-apoptotic repertoire in these cells. In this study we suggest that a likely
mechanism of decreased viability in TC71 cells treated with IGF-1R inhibitors is partially
due to apoptosis.
Cytoplasmic blebbing was observed in cells treated with PPP and IMC-A12,
leading us to suspect that these cells were apoptotic. Our suspicions were sustained by
the observation of PARP cleavage in these cells. It is important to mention that blebbing
and PARP cleavage are not exclusive to apoptosis. Cleaved PARP is present in a variety
of cell death processes including apoptosis, necrosis, and autophagy[29]. Further
experiments are needed to confirm the role of apoptosis in the observed decrease in
viable cell number. Further experiments including annexin V staining, cleaved caspase
assays, and TUNEL staining are needed to verify apoptosis as the mechanism of cell
death.
The finding that BMI-1 knockdown cells treated with PPP displayed higher
cleaved PARP levels than either untreated BMI-1 knockdown cells or BMI-1-positive
cells demonstrates that BMI-1 and IGF-1 do in fact cooperate to promote survival in
ESFT.
The hypothesis that BMI-1 and IGF-1 signaling converges on the PI3K/Akt
pathway in the promotion of survival in ESFT explains the observed phenomenon
displayed in the PARP cleavage assay. Previous works have demonstrated that both IGF-
1 signaling and BMI-1 expression promote Akt activation through phosphorylation [21].
27
27
While the molecular mechanism of BMI-1’s promotion of Akt phosphorylation is yet
unknown in ESFT, the effect of IGF-1 signaling on the PI3K/Akt pathway have been
described in detail. BMI-1 has been shown to repress expression of the tumor suppressor
PTEN in nasopharyngeal cancer [21], however the multifaceted nature of BMI-1’s effects
in cancer initiation and progression are still being elucidated.
In this paper I have presented evidence supporting my hypothesis that the
interaction of BMI-1 and IGF-1 signaling converges on the PI3K/Akt pathway. The
observation that levels of pAkt are markedly decreased in cells treated with siRNA-
mediated BMI-1 knockdown and PPP compared to cells treated with either condition
alone provides a clear point of convergence. While knocking down BMI-1 expression
alone did not significantly effect pAkt levels; and treatment with the IGF-1R inhibitor
PPP alone yielded the same results, combining the effects of BMI-1 knockdown and PPP
treatment significantly decreased the amount of phosphorylated Akt in these cells.
The observations that TC71 cells expressing low levels of BMI-1 show increased
sensitivity to IGF-1R blockade; that anti-IGF-1R therapy in combination with BMI-1
knockdown further decreases viable cell number likely through apoptosis; and that the
combined BMI-1 knockdown and PPP-treated cells display significantly decreased levels
of pAkt, have allowed me to put forward a model of interaction between IGF-1 and BMI-
1 (Figs. 9A-9D).
I propose that BMI-1 and IGF-1 signaling are two branches of a survival pathway
converging on activation of Akt to promote survival of ESFT cells. IGF-1 signaling
drives Akt phosphorylation, promoting cell cycle entry, metabolism, and survival.
28
28
Similarly, BMI-1 drives Akt phosphorylation through a mechanism that has not yet been
elucidated. By removing either the BMI-1 or IGF-1 signaling branch, the other is
sufficient to accommodate the loss and Akt phosphorylation is not affected. However, by
removing BMI-1 and subjecting ESFT cells to IGF-1R blockade thereby removing IGF-1
signaling, cells are unable to maintain Akt phosphorylation, resulting in cell death by
apoptosis.
In summary, we have shown that BMI-1 and IGF-1 signaling act together to
support survival of ESFT cells. The molecular mechanisms of BMI-1 in promoting a
stem cell phenotype in both normal tissues and human cancers are likely to be complex
involving multiple cellular processes. Even so, our findings support the hypothesis that
BMI-1 acts in conjunction with IGF-1 signaling in promoting ESFT tumorigenesis
through activation of the PI3K/Akt survival pathway. Future studies aimed at
understanding the molecular mechanisms of BMI-1 in supporting and supplementing the
IGF-1/PI3K/Akt signaling pathway may not only improve our understanding of ESFT
biology, but also improve therapy by identifying candidates likely to benefit from novel
molecular therapies.
29
29
Figures 9A-9D. Proposed model of interaction between BMI-1 and IGF-1 signaling.
A. BMI-1 and the IGF-1 signaling pathway promote phosphorylation and activation of
Akt resulting in survival, cell cycle progression, metabolism, and angiogenesis. B. With
the removal of IGF-1 signaling via treatment with IGF-1R inhibitors PPP and IMC-A12
(treated control), BMI-1 is sufficient in promoting phosphorylation and activation of Akt
resulting in survival, cell cycle progression, metabolism, and angiogenesis. C. Amid
knockdown of BMI-1 through siRNA-mediated suppression (untreated knockdown),
IGF-1 signaling is sufficient in promoting phosphorylation and activation of Akt resulting
in survival, cell cycle progression, metabolism, and angiogenesis. D. Cells treated with
BMI-1 knockdown in conjunction with removal of IGF-1 signaling (treated knockdown)
are unable to maintain Akt phosphorylation, resulting in cell death by apoptosis.
30
30
References
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that originates from a primitive hematopoietic cell. Nat Med, 1997. 3(7): p. 730-7.
2. Esiashvili, N., M. Goodman, and R.B. Marcus, Jr., Changes in incidence and survival
of Ewing sarcoma patients over the past 3 decades: Surveillance Epidemiology and End
Results data. J Pediatr Hematol Oncol, 2008. 30(6): p. 425-30.
3. Balamuth, N.J. and R.B. Womer, Ewing's sarcoma. Lancet Oncol, 2010. 11(2): p. 184-
92.
4. Grier, H.E., The Ewing family of tumors. Ewing's sarcoma and primitive
neuroectodermal tumors. Pediatr Clin North Am, 1997. 44(4): p. 991-1004.
5. Rosen, G., et al., Proceedings: Disease-free survival in children with Ewing's sarcoma
treated with radiation therapy and adjuvant four-drug sequential chemotherapy. Cancer,
1974. 33(2): p. 384-93.
6. Ordonez, J.L., et al., Advances in Ewing's sarcoma research: where are we now and
what lies ahead? Cancer Res, 2009. 69(18): p. 7140-50.
7. Zha, J. and M.R. Lackner, Targeting the insulin-like growth factor receptor-1R
pathway for cancer therapy. Clin Cancer Res, 2010. 16(9): p. 2512-7.
8. Grinstein, E. and P. Wernet, Cellular signaling in normal and cancerous stem cells.
Cell Signal, 2007. 19(12): p. 2428-33.
9. Reya, T., et al., Stem cells, cancer, and cancer stem cells. Nature, 2001. 414(6859): p.
105-11.
10. Klein, S., F. McCormick, and A. Levitzki, Killing time for cancer cells. Nat Rev
Cancer, 2005. 5(7): p. 573-80.
11. Clarke, M.F. and M. Fuller, Stem cells and cancer: two faces of eve. Cell, 2006.
124(6): p. 1111-5.
12. Lessard, J. and G. Sauvageau, Bmi-1 determines the proliferative capacity of normal
and leukaemic stem cells. Nature, 2003. 423(6937): p. 255-60.
13. Beachy, P.A., S.S. Karhadkar, and D.M. Berman, Tissue repair and stem cell
renewal in carcinogenesis. Nature, 2004. 432(7015): p. 324-31.
31
31
14. Buszczak, M. and A.C. Spradling, Searching chromatin for stem cell identity. Cell,
2006. 125(2): p. 233-6.
15. Valk-Lingbeek, M.E., S.W. Bruggeman, and M. van Lohuizen, Stem cells and
cancer; the polycomb connection. Cell, 2004. 118(4): p. 409-18.
16. Park, I.K., et al., Bmi-1 is required for maintenance of adult self-renewing
haematopoietic stem cells. Nature, 2003. 423(6937): p. 302-5.
17. Douglas, D., et al., BMI-1 promotes ewing sarcoma tumorigenicity independent of
dCDKN2A repression. Cancer Res, 2008. 68(16): p. 6507-15.
18. Leung, C., et al., Bmi1 is essential for cerebellar development and is overexpressed
in human medulloblastomas. Nature, 2004. 428(6980): p. 337-41.
19. Hemmati, H.D., et al., Cancerous stem cells can arise from pediatric brain tumors.
Proc Natl Acad Sci U S A, 2003. 100(25): p. 15178-83.
20. Cui, H., et al., Bmi-1 is essential for the tumorigenicity of neuroblastoma cells. Am J
Pathol, 2007. 170(4): p. 1370-8.
21. Song, L.B., et al., The polycomb group protein Bmi-1 represses the tumor suppressor
PTEN and induces epithelial-mesenchymal transition in human nasopharyngeal
epithelial cells. J Clin Invest, 2009. 119(12): p. 3626-36.
22. Cooper, A., et al., Ewing tumors that do not over-express BMI-1 are a distinct
molecular subclass with variant biology: A report from the Children's Oncology Group.
Clin Cancer Res, 2011. 17(1): p. 56-66.
23. Gualberto, A. and M. Pollak, Emerging role of insulin-like growth factor receptor
inhibitors in oncology: early clinical trial results and future directions. Oncogene, 2009.
28(34): p. 3009-21.
24. Gualberto, A. and D.D. Karp, Development of the monoclonal antibody figitumumab,
targeting the insulin-like growth factor-1 receptor, for the treatment of patients with non-
small-cell lung cancer. Clin Lung Cancer, 2009. 10(4): p. 273-80.
25. Carden, C.P., L.R. Molife, and J.S. de Bono, Predictive biomarkers for targeting
insulin-like growth factor-I (IGF-I) receptor. Mol Cancer Ther, 2009. 8(8): p. 2077-8.
26. Zha, J., et al., Molecular predictors of response to a humanized anti-insulin-like
growth factor-I receptor monoclonal antibody in breast and colorectal cancer. Mol
Cancer Ther, 2009. 8(8): p. 2110-21.
32
32
27. Girnita, A., et al., Cyclolignans as inhibitors of the insulin-like growth factor-1
receptor and malignant cell growth. Cancer Res, 2004. 64(1): p. 236-42.
28. Yin, S., et al., Targeting the insulin-like growth factor-1 receptor by
picropodophyllin as a treatment option for glioblastoma. Neuro Oncol, 2010. 12(1): p.
19-27.
29. Chaitanya, G.V. and P.P. Babu, Differential PARP cleavage: an indication of
heterogeneous forms of cell death and involvement of multiple proteases in the infarct of
focal cerebral ischemia in rat. Cell Mol Neurobiol, 2009. 29(4): p. 563-73.
30. Van Lohuizen, M., et al., Identification of cooperating oncogenes in E mu-myc
transgenic mice by provirus tagging. Cell, 1991. 65: p. 737-52.
31. Haupt, Y., et al., Novel zinc finger gene implicated as myc collaborator by
retrovirally accelerated lymphomagenesis in E mu-myctransgenic mice. Cell, 1991. 65: p.
753-63.
32. Van der Lugt, N.M., et al., Posterior transformation, neurological, abnormalities,
and severe hematopoietic defects in mice with a targeted deletion of the bmi-1 proto-
oncogene. Genes Dev, 1994. 8: p. 757-69.
33. Jiang, L., et al., Bmi-1, stem cells and cancer. ABBS, 2009. 41(07): p. 527-34.
34. Alkema, M.J., et al., Characterization and chromosomal localization of the human
protooncogene Bmi-1. Hum Mol Genet, 1993. 10: p. 1597-1603.
35. Alkema, M.J., et al., Identification of Bmi1-interacting proteins as constituents of a
multimeric mammalian polycomb complex. Genes Dev, 1997. 11: p. 226-40.
36. Itahana, K., et al., Control of the replicative life span of human fibroblasts by p16
and the polycomb protein Bmi-1. Mol Cell Biol, 2003. 23: p. 389-401.
37. Lessard, J., et al., Bmi-1 determines the proliferative capacity of normal and
leukaemic stem cells. Nature, 2003. 423: p. 255-60.
38. Park, I.K., et al., Bmi-1 is required for maintenance of adult self-renewing
haematopoietic stem cells. Nature, 2003. 423: p. 302-305.
39. Sawa, M., et al., BMI-1 is highly expressed in M0-subtype acute myeloid leukemia.
Int J Hematol, 2005. 82: p. 42-47.
33
33
40. Yang, J., et al., Bmi-1 is a target gene for SALL4 in hematopoietic and leukemic
cells. Proc Natl Acad Sci USA, 2007. 104: p. 10494-99.
41. Raaphorst, F.M., Self-renewal of hematopoietic and leukemic stem cells: a central
role for the Polycomb-group gene Bmi-1. Trends Immulol, 2003. 24: p. 522-24.
42. Song, L.B., et al., Bmi-1 is a novel molecular marker of nasopharyngeal carcinoma
progression and immortalizes primary human nasopharyngeal epithelial cells. Cancer
Res, 2006. 66: p. 6225-32.
43. Dimri, G.P., et al., The Bmi-1 oncogene induces telomerase activity and immortalizes
human mammary epithelial cells. Cancer Res, 2002. 62: p. 4736-45.
44. Cui, H., et al., Bmi-1 is essential for the tumorigenicity of neuroblastoma cells. Am J
Pathol, 2007. 170: p. 1370-78.
45. Jacobs, J.J., et al., Bmi-1 collaborates with c-Myc in tumorigenesis by inhibiting c-
Myc-induced apoptosis via INK4a/ARF. Genes Dev, 1999. 13: p. 2678-90.
46. Bartkova, J., et al., The p16-cyclin D/cdk4-pRb pathway as a functional unit
frequently altered in melanoma pathogenesis. Cancer Res, 1996. 56: p. 5475-83.
47. Nilsson, K. and Landberg, G., Subcellular localization, modification and protein
complex formation of the cdk-inhibitor p16 in Rb-functional and Rb-inactivated tumor
cells. Int J Cancer, 2006. 118: p. 1120-25.
48. Grinstein, E. and Wernet, P., Cellular signaling in normal and cancerous stem cells.
Cell Signal, 2007. 19: p. 2428-33.
49. Zhang, Y., et al., ARF promotes MDM2 degradation and stablizes p53: ARF-INK4a
locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell, 1998. 92:
p. 725-34.
50. Hopkins, A., Crowe, P.J., and Yang, J.L., Effect of type 1 insulin-like growth factor
receptor targeted therapy on chemotherapy in human cancer and the mechanisms
involved. J Cancer Res Clin Oncol, 2010. 136: p. 639-50.
34
34
Bibliography
Alkema, M.J., et al., Characterization and chromosomal localization of the human
protooncogene Bmi-1. Hum Mol Genet, 1993. 10: p. 1597-1603.
Alkema, M.J., et al., Identification of Bmi1-interacting proteins as constituents of a
multimeric mammalian polycomb complex. Genes Dev, 1997. 11: p. 226-40.
Balamuth, N.J. and R.B. Womer, Ewing's sarcoma. Lancet Oncol, 2010. 11(2): p. 184-
92.
Bartkova, J., et al., The p16-cyclin D/cdk4-pRb pathway as a functional unit frequently
altered in melanoma pathogenesis. Cancer Res, 1996. 56: p. 5475-83.
Beachy, P.A., S.S. Karhadkar, and D.M. Berman, Tissue repair and stem cell renewal in
carcinogenesis. Nature, 2004. 432(7015): p. 324-31.
Bonnet, D. and J.E. Dick, Human acute myeloid leukemia is organized as a hierarchy
that originates from a primitive hematopoietic cell. Nat Med, 1997. 3(7): p. 730-
7.
Buszczak, M. and A.C. Spradling, Searching chromatin for stem cell identity. Cell, 2006.
125(2): p. 233-6.
Carden, C.P., L.R. Molife, and J.S. de Bono, Predictive biomarkers for targeting insulin-
like growth factor-I (IGF-I) receptor. Mol Cancer Ther, 2009. 8(8): p. 2077-8.
Chaitanya, G.V. and P.P. Babu, Differential PARP cleavage: an indication of
heterogeneous forms of cell death and involvement of multiple proteases in the
infarct of focal cerebral ischemia in rat. Cell Mol Neurobiol, 2009. 29(4): p. 563-
73.
Clarke, M.F. and M. Fuller, Stem cells and cancer: two faces of eve. Cell, 2006. 124(6):
p. 1111-5.
Cooper, A., et al., Ewing tumors that do not over-express BMI-1 are a distinct molecular
subclass with variant biology: A report from the Children's Oncology Group. Clin
Cancer Res, 2011. 17(1): p. 56-66.
Cui, H., et al., Bmi-1 is essential for the tumorigenicity of neuroblastoma cells. Am J
Pathol, 2007. 170(4): p. 1370-8.
35
35
Dimri, G.P., et al., The Bmi-1 oncogene induces telomerase activity and immortalizes
human mammary epithelial cells. Cancer Res, 2002. 62: p. 4736-45.
Douglas, D., et al., BMI-1 promotes ewing sarcoma tumorigenicity independent of
dCDKN2A repression. Cancer Res, 2008. 68(16): p. 6507-15.
Esiashvili, N., M. Goodman, and R.B. Marcus, Jr., Changes in incidence and survival of
Ewing sarcoma patients over the past 3 decades: Surveillance Epidemiology and
End Results data. J Pediatr Hematol Oncol, 2008. 30(6): p. 425-30.
Girnita, A., et al., Cyclolignans as inhibitors of the insulin-like growth factor-1 receptor
and malignant cell growth. Cancer Res, 2004. 64(1): p. 236-42.
Grier, H.E., The Ewing family of tumors. Ewing's sarcoma and primitive
neuroectodermal tumors. Pediatr Clin North Am, 1997. 44(4): p. 991-1004.
Grinstein, E. and P. Wernet, Cellular signaling in normal and cancerous stem cells. Cell
Signal, 2007. 19(12): p. 2428-33.
Gualberto, A. and D.D. Karp, Development of the monoclonal antibody figitumumab,
targeting the insulin-like growth factor-1 receptor, for the treatment of patients
with non-small-cell lung cancer. Clin Lung Cancer, 2009. 10(4): p. 273-80.
Gualberto, A. and M. Pollak, Emerging role of insulin-like growth factor receptor
inhibitors in oncology: early clinical trial results and future directions.
Oncogene, 2009. 28(34): p. 3009-21.
Haupt, Y., et al., Novel zinc finger gene implicated as myc collaborator by retrovirally
accelerated lymphomagenesis in E mu-myctransgenic mice. Cell, 1991. 65: p.
753-63.
Hemmati, H.D., et al., Cancerous stem cells can arise from pediatric brain tumors. Proc
Natl Acad Sci U S A, 2003. 100(25): p. 15178-83.
Hopkins, A., Crowe, P.J., and Yang, J.L., Effect of type 1 insulin-like growth factor
receptor targeted therapy on chemotherapy in human cancer and the mechanisms
involved. J Cancer Res Clin Oncol, 2010. 136: p. 639-50.
Itahana, K., et al., Control of the replicative life span of human fibroblasts by p16 and the
polycomb protein Bmi-1. Mol Cell Biol, 2003. 23: p. 389-401.
Jacobs, J.J., et al., Bmi-1 collaborates with c-Myc in tumorigenesis by inhibiting c-Myc-
induced apoptosis via INK4a/ARF. Genes Dev, 1999. 13: p. 2678-90.
36
36
Jiang, L., et al., Bmi-1, stem cells and cancer. ABBS, 2009. 41(07): p. 527-34.
Klein, S., F. McCormick, and A. Levitzki, Killing time for cancer cells. Nat Rev Cancer,
2005. 5(7): p. 573-80.
Lessard, J. and G. Sauvageau, Bmi-1 determines the proliferative capacity of normal and
leukaemic stem cells. Nature, 2003. 423(6937): p. 255-60.
Lessard, J., et al., Bmi-1 determines the proliferative capacity of normal and leukaemic
stem cells. Nature, 2003. 423: p. 255-60.
Leung, C., et al., Bmi1 is essential for cerebellar development and is overexpressed in
human medulloblastomas. Nature, 2004. 428(6980): p. 337-41.
Nilsson, K. and Landberg, G., Subcellular localization, modification and protein complex
formation of the cdk-inhibitor p16 in Rb-functional and Rb-inactivated tumor
cells. Int J Cancer, 2006. 118: p. 1120-25.
Ordonez, J.L., et al., Advances in Ewing's sarcoma research: where are we now and what
lies ahead? Cancer Res, 2009. 69(18): p. 7140-50.
Park, I.K., et al., Bmi-1 is required for maintenance of adult self-renewing
haematopoietic stem cells. Nature, 2003. 423(6937): p. 302-5.
Raaphorst, F.M., Self-renewal of hematopoietic and leukemic stem cells: a central role
for the Polycomb-group gene Bmi-1. Trends Immulol, 2003. 24: p. 522-24.
Reya, T., et al., Stem cells, cancer, and cancer stem cells. Nature, 2001. 414(6859): p.
105-11.
Rosen, G., et al., Proceedings: Disease-free survival in children with Ewing's sarcoma
treated with radiation therapy and adjuvant four-drug sequential chemotherapy.
Cancer, 1974. 33(2): p. 384-93.
Sawa, M., et al., BMI-1 is highly expressed in M0-subtype acute myeloid leukemia. Int J
Hematol, 2005. 82: p. 42-47.
Song, L.B., et al., Bmi-1 is a novel molecular marker of nasopharyngeal carcinoma
progression and immortalizes primary human nasopharyngeal epithelial cells.
Cancer Res, 2006. 66: p. 6225-32.
37
37
Song, L.B., et al., The polycomb group protein Bmi-1 represses the tumor suppressor
PTEN and induces epithelial-mesenchymal transition in human nasopharyngeal
epithelial cells. J Clin Invest, 2009. 119(12): p. 3626-36.
Valk-Lingbeek, M.E., S.W. Bruggeman, and M. van Lohuizen, Stem cells and cancer;
the polycomb connection. Cell, 2004. 118(4): p. 409-18.
Van der Lugt, N.M., et al., Posterior transformation, neurological, abnormalities, and
severe hematopoietic defects in mice with a targeted deletion of the bmi-1 proto-
oncogene. Genes Dev, 1994. 8: p. 757-69.
Van Lohuizen, M., et al., Identification of cooperating oncogenes in E mu-myc
transgenic mice by provirus tagging. Cell, 1991. 65: p. 737-52.
Yang, J., et al., Bmi-1 is a target gene for SALL4 in hematopoietic and leukemic cells.
Proc Natl Acad Sci USA, 2007. 104: p. 10494-99.
Yin, S., et al., Targeting the insulin-like growth factor-1 receptor by picropodophyllin as
a treatment option for glioblastoma. Neuro Oncol, 2010. 12(1): p. 19-27.
Zha, J. and M.R. Lackner, Targeting the insulin-like growth factor receptor-1R pathway
for cancer therapy. Clin Cancer Res, 2010. 16(9): p. 2512-7.
Zha, J., et al., Molecular predictors of response to a humanized anti-insulin-like growth
factor-I receptor monoclonal antibody in breast and colorectal cancer. Mol
Cancer Ther, 2009. 8(8): p. 2110-21.
Zhang, Y., et al., ARF promotes MDM2 degradation and stablizes p53: ARF-INK4a
locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell,
1998. 92: p. 725-34.
Abstract (if available)
Abstract
Purpose: Ewing sarcoma family tumors (ESFT) are highly aggressive pediatric and young adult stem cell tumors for which prognostic biomarkers and novel treatments are needed. New treatments targeting the IGF-1R signaling pathway show promise, but may not be effective in treating all patients. We have assessed the potential clinical significance of BMI-1 expression level on treatments utilizing IGF-1R inhibition.
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Creator
Russell, Darren Jason
(author)
Core Title
Investigating the roles of BMI-1 and IGF-1 in promoting ESFT survival
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Experimental and Molecular Pathology
Publication Date
01/19/2011
Defense Date
07/21/2010
Publisher
University of Southern California
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BMI-1, IFG-1,cancer,ESFT,Ewing's sarcoma,IGF-1R,OAI-PMH Harvest
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Erdreich-Epstein, Anat (
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), Hofman, Florence M. (
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), Roy-Burman, Pradip (
committee member
), Widelitz, Randall B. (
committee member
)
Creator Email
darrussell@gmail.com,djrussel@usc.edu
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Tags
BMI-1, IFG-1
ESFT
Ewing's sarcoma
IGF-1R