Investigating the Molecular Regulation
of Brain Tumor Malignancy
Patrick M. Martin, Ph. D.
University of North Texas Health Sciences Center
Department of Biology
September 27, 2010
Primary Tumors of the Brain


Account for 2% of all primary tumors
World Health Organization classifies
brain tumors into four grades




Grade I: Pilocytic astrocytoma
Grade II: Diffuse astrocytoma
Grade III: Anaplastic astrocytoma
(Diffuse)
Grade IV: Glioblastoma
Multiforme
adapted from: Reifenberger and Collins (2004)
Glioblastoma Multiforme (GBM)

The most lethal
and malignant
form of primary
brain tumors
found in adults

Prognosis for
Patients is quite
poor



Incidence rate of 3.1
per 100,000
persons/year in
United States
2-year survival rate
of 8%
Median survival Rate
of 13 months
Chromosomal and genetic alterations
associated with astrocytic gliomas
Astrocyte or glial precursor cells
 EGFR amplification/overexpression
 P53 mutation (<30%)
 RB1 mutation/homozygous deletion
 LOH 10p and 10q
 CDK 4 and 6
 PTEN mutation (30-40%)
Primary GBM (WHO Grade IV)
Adapted from Kleihus et al. (2000)
Glioblastoma Growth Characteristics
● Rapid growth and diffuse infiltration into normal
brain
● GBM is often resistant to radiotherapy and many
chemotherapeutic agents are not effective
● Transcription Factors (Fra-1, C-Jun, C-Fos, etc.),
Adhesion molecules (CD44, Integrins, etc.)are
constitutively active or over-expressed in tumor
samples and GBM cell lines
More GBM Images
MRI of GBM in 55
year old Female.
British Journal of
Radiology
GBM at time of surgical resection
American Journal of Neuroradiology
GBM at autopsy (notice secondary tumor site),WebPath
Notable Individuals Diagnosed
with Brain Tumors
Senator Edward “Ted” Kennedy
Johnnie Cochran
Glioblastoma Migration and Invasion
Claes A et al. Acta Neuropathologica 2007;114(5):443-458
CD44: An Adhesion Molecule

A transmembrane glycoprotein

CD44 is a membrane glycoprotein that is the
principal membrane receptor for hyaluronan (HA).

HA is a major component of the extra cellular
matrix (ECM) in the brain
Role of CD44 in Gliomagenesis

CD44 and HA expression is significantly
increased during the formation and migration of
GBMs (reviewed by Bellail et al., 2004)

Expression in primary malignant gliomas is
restricted to the white matter (Bellail et al.,
2004)
Brain Sections
Role of Fra-1

Fos Related Antigen

Transcription factor that is a member of the
activator protein-1 (AP-1) family

Early response proto-oncogene

Response to growth factors and promote
increased transcription of specific gene
targets (Curran & Franza, 1988)

Expression and activity are increased in
high grade gliomas especially
glioblastomas (Debinski & Gibo 2005)
Real-time Q-PCR for quantitation of selected genes up-regulated in (A) asbestos-exposed RPM
cells and (B) mesothelioma 23 after transfection with sifra-1 or sd.
Ramos-Nino M E et al. Cancer Res 2003;63:3539-3545
Use of shRNA constructs to knock-down Fra-1 confirm Fra-1-dependent CD44 expression in MMs. A. Western
blots of SV40- (MM1) and SV40+ (MM3) lines showing empty vector (EV) controls or RNAi shFra-1
constructs. B. Immunofluorescence image showing CD44 (red) levels MM cell lines.
Ramos-Nino et al. Molecular Cancer 2007;6:81
Overarching Research Objective
To investigate the regulation of CD44
expression in human glioma cells by
the transcription factor Fra-1


Approach #1:


Approach #2:


To examine the expression levels of CD44 and
Fra-1 in glioma cells
To assay whether Fra-1 promotes CD44
expression in GBM cells
Approach #3:

To investigate whether CD44-mediated glioma
cell adhesion is promoted by the expression of
Fra-1
Overall Hypothetical Model
Malignant Gliomas
Cell Invasive Migration
Research Aim 1

To examine the expression levels
of CD44 and Fra-1 in glioma cells

Hypothesis:


CD44 expression levels will correlate with the
levels of Fra-1 expression in each cell line
Experimental Design:

Stimulation of GBM cells with Growth Factor
■ Epidermal Growth Factor (EGF)
■ Hepatocyte Growth Factor (HGF)


Western Blot Analysis
Immunofluorescence
Hypothetical Model
Growth Factor
Fra-1
Fra-1
Fra-1
Fra-1
Fra-1
Preliminary Study of the Cell
Lines
EGF & HGF Stimulation of U-251 MG
U-251 MG
EGF (20 ng/ml)
HGF (50 ng/ml)
CD-44
80 kDa
Rel. Abs.
1.0
0.9
1.0
1.0
1.6
1.0
Fra-1
45 kDa
Rel. Abs.
1.0
1.3
0.9
1.0
1.8
1.1
β - Tubulin
EGF & HGF Stimulation of U-1242 MG
U-1242 MG
EGF (20 ng/ml)
HGF (50 ng/ml)
CD-44
80 kDa
Rel. Abs.
1.0
1.1
1.4
1.0
1.0
1.0
Fra-1
45 kDa
Rel. Abs.
1.0
1.6
3.7
1.0
2.2
1.4
β - Tubulin
Localization of CD44 and
Fra-1 in Human GBM Cell
lines
EGF Stimulation of U-251 MG
HGF Stimulation of U-251 MG
EGF Stimulation of U-1242 MG
HGF Stimulation of U-1242 MG
Preliminary Conclusions # 1

U-251 MG



U-1242 MG



EGF does not increase CD44 expression
HGF stimulation does increase CD44 expression
EGF stimulation does increase CD44 expression
HGF stimulation does not increase CD44
expression
HGF increases Fra-1 expression more
dramatically then EGF stimulation


HGF – 4 hour stimulation
EGF – 24 hour stimulation
Research Aim 2

To determine whether the expression
of CD44 is regulated by Fra-1
expression in glioma cells

Hypothesis:


Silencing the expression of Fra-1 will decrease the
expression of CD44
Experimental Design:



RNA Interference via Nucleofection
Western Blot Analysis
Immunofluorescence
Hypothetical Model
Growth Factor
Fra-1
siRNA
Fra-1
Fra-1
Fra-1
Fra-1
Fra-1
fra-1-siRNA treated Human
GBM cells
EGF stimulation of fra-1-siRNA treated U-251 GBM cells
U-251 MG
EGF
(20 ng/ml)
CD-44
80 kDa
Rel. Abs.
1.0
0.9
1.0
1.0
0.9
0.7
Fra-1
45 kDa
Rel. Abs.
1.0
1.1
1.4
0.9
1.1
0.9
β - Tubulin
HGF stimulation of fra-1-siRNA treated U-251 GBM cells
U-251 MG
HGF
(50 ng/ml)
CD-44
80 kDa
Rel. Abs.
1.0
1.7
1.9
2.3
1.9
2.1
Fra-1
45 kDa
Rel. Abs.
1.0
1.3
1.4
1.4
1.2
1.4
β - Tubulin
EGF stimulation of fra-1-siRNA treated U-1242 GBM cells
U-1242 MG
EGF
(20 ng/ml)
CD-44
80 kDa
Rel. Abs.
1.0
1.1
1.4
1.3
1.1
1.0
Fra-1
45 kDa
Rel. Abs.
1.0
1.6
1.7
1.4
1.3
1.1
β - Tubulin
HGF stimulation of fra-1-siRNA treated U-1242 GBM cells
U-1242 MG
HGF
(50 ng/ml)
CD-44
80 kDa
Rel. Abs.
1.0
0.8
1.2
0.5
0.8
1.1
Fra-1
45 kDa
Rel. Abs.
1.0
1.7
1.8
0.6
1.3
1.5
β - Tubulin
Localization of CD44 and Fra-1
of fra-1-siRNA treated U-1242
MG cells
EGF stimulation of fra-1-siRNA treated U-1242 GBM cells
24 hr
Control siRNA
Fra-1 siRNA
HGF stimulation of fra-1-siRNA treated U-1242 GBM cells
24 hr
Control siRNA
Fra-1 siRNA
Preliminary Conclusions #2

EGF stimulation after fra-1-siRNA
decreases CD44 and Fra-1 expression

HGF stimulation after fra-1-siRNA has
no affect on CD44 and Fra-1
expression

HGF demonstrates a greater
regulatory role on Fra-1 expression
than EGF
Research Aim 3

To investigate whether CD44-mediated
glioma cell adhesion is promoted by
the expression of Fra-1

Hypothesis:


Glioma cell adhesion is promoted by the
activation of Fra-1 through the regulation of
CD44 expression
Experimental Design:


RNA Interference
Cell Adhesion Assay
Methods: Adhesion Assay
Adhesion Assay of Human
GBM Cell lines
Adhesion Assay of EGF and HGF
stimulated U-251 MG Cell
Adhesion Assay of EGF and HGF
stimulated U-1242 MG Cell
Adhesion Assay of siRNA
treated Human GBM Cell lines
Adhesion Assay of siRNA treated EGF
stimulated U-251 MG Cell
Adhesion Assay of siRNA treated HGF
stimulated U-251 MG Cell
Adhesion Assay of siRNA treated EGF
stimulated U-1242 MG Cell
Adhesion Assay of siRNA treated HGF
stimulated U-1242 MG Cell
Preliminary Conclusions #3

Cell lines demonstrated an increase
adherence to HA after growth factor
stimulation


U-251 MG – 4 hour stimulation
U-1242 MG – 24 hour stimulation

Data is comparable to the western blot
analysis of the GBM cells

CD44 binding to HA is cell line specific

CD44-mediated HA binding of GBM cells can
be regulated by Fra-1 expression
Overall Summary

Confirmed CD44 and Fra-1 expression in
GBM Cell lines (U-1242 MG and U-251 MG)

The regulation of CD44 is linked to Fra-1
expression and/or activity

CD44-mediated HA binding of GBM cells is
regulated by Fra-1 expression

EGF and HGF demonstrate a differential
regulatory effect on Fra-1 and CD44
expression in U-251 MG and U-1242 MG cell
lines
Final Model
WHY?
Theory of HGF regulation of Fra-1

Explanation # 1:


Protective resistance against the fra-1
siRNA
Explanation # 2:

Fra-1 can autoregulate the transcription
of its own transcript
Theory of HGF regulation of Fra-1

HGF increases Fra-1 expression
more dramatically then EGF
stimulation


HGF – 4 hour stimulation
EGF – 24 hour stimulation
Theory of HGF regulation of Fra-1

Explanation # 1:


Protective resistance against the fra-1
siRNA
Explanation # 2:

Fra-1 can autoregulate the transcription
of its own transcript
Proposed Model of Fra-1 Autoregulation
fra-1
siRNA
HGF stimulation of fra-1-siRNA treated Human
GBM Cell lines
Future Directions of Study

Migration Assays

Flow Cytometry of Human GBM cell
lines

Characterize the Growth Factor receptors

Real Time PCR

Inhibitor Studies
Acknowledgements
Martin Lab
Patrice Cagle
Timothy Raines (UVA PhD program)
Tyler Smith
Khrystal McGrant
Wake Forest University
Dr. Waldemar Debinski
Denise Gibo
Dr. Carla Lema-Tome’
Funding Agencies
NCI Grant #: NIH-NCI 1P20CA138020-01 (PMM)
RIMI Grant#: NIH-NIGMS 5P20MD000546-07 (PMM)
NCATSU College of Arts and Sciences (PMM)
MERCK/AAAS (PMM)
MARC and RISE (PMM)
Waste Management (TAR)
North Carolina Alliance to Create Opportunity Through Education (NC OPT-ED)
(TAR and KMG)
THANK YOU