2E Conférence Québécoise Sur La Résistance Thérapeutique du Cancer
Q-CROC
Montréal, November 5-6, 2010
Potential Conflict of Interest
• Dr. Thierry Muanza
– None
Radiotherapy

Along with surgery and chemotherapy, radiotherapy is a
mainstay of cancer treatment

75% cancer patients in the course of their disease will
undergo radiotherapy
Radiobiology

IR local release of large amount of energy

~ 33eV dissipated / ionizing event, enough to break strong
chemical bond


energy associated C=C bond is 4.9 eV
Types:


Electromagnetic
particulate
IR: mechanism of action
.
Radiation Induced DNA Damage
http://www.radiation-scott.org/radsource
Chromosomal
Damage
Apoptosis
Reproductive
death
Necrosis
Radiation Induced DNA Damage
G1 arrest
pH2XA
E2F
E2F
ATM
P53
pRb
P53
pRb
Cycline B
BAX
cdK /Cycline D
Cycline E
p21 WAF1 / CIPI
N Cycline-B-p34 cdc2
Cytochrome c
Apaf1
Smac/
Diablo
Survivin
G2 arrest
Caspase 9
Caspase 3
Caspase 6
Apoptosis
DNA Repair Mechanisms
RNA Pol II or XPC
HR
Rad51 /Rad52
XPD / XPB
NER
pH2aX
ERCC1 / XPG
SSB
DNA Pol  / ligase
BRAC1/BRAC2
ATM
BER
Rad52/MRE11/NBSI
DSB
NHEJ
Completion
of DSB repair
Completion of repair
XRCC1
DNA-PKcs / Ku80/70
MRE11/Rad51/NBSI
DNA Pol  / DNA ligase III
DNA ligase IV, XRCC4
Cell Survival Curves
CA: RT sensitivity
Cell cycle: RT sensitivity
Tumor Oxygenation
Re-oxygenation
Effect of Oxygen
RT resistance
Ma et al. JCO 21, 2003
Radiosensitizers
Radiosensitizers

Nonhypoxic

Halogenated pyrimidines



Hypoxic




DNA
Cycling tissues
Free radical process (fixed)
Misonidazole (toxicity)
Overgaard metaanalyisis: 4.6% LC, 2.8% OS
Hypoxic cytotoxins

Bioreductive Rx


Tirapazamine (nitroxide)
Mitomycin C
Radiosensitizers
Radiosensitizers
Cell Proliferation Assay (MTT)
MDA-MB-468 cells
110
ZRBA1 with Radiation
100
Iressa with Radiation
90
ZRBA1
Percent Control Cell Viability
80
Iressa
70
60
50
40
30
20
10
0
0
10
20
30
40
50
60
70
80
90
100
110
Drug Concentration (uM)
Heravi, Muanza et al. ACD 20, 2009
Cell Proliferation Assay (MTT)
Control
Percent cell viability
140
120
100
80
p<0.0049
ZRBA1
**
60
**
40
20
0
No XRT
RX then XRT
RX+XRT
XRT then RX
**: P<0.01
Colony Forming Assay
1
ZRBA1
Iressa
Survival Fraction
0.1
Control
Radiation Dose (Gy)
2
4
6
0.01
0.001
0.0001
0
2
4
6
Radiation Dose (Gy)
8
10
DER
1.6
2.2
2.9
γH2AX Immunofluorescent Staining
Control
1 Gy
0.5 Gy
γH2AX Immunofluorescent Staining
Control
ZRBA1 18uM
ZRBA1 9uM
γH2AX Immunofluorescent Staining
Control
ZRBA1 9uM
0.5 Gy
0.5 Gy and ZRBA1 9uM
γH2AX Immunofluorescent Staining
Control
ZRBA1 9uM
1 Gy
1 Gy and ZRBA1 9uM
γH2AX Immunofluorescent Staining
Control
9 uMZRBA1
18 uM ZRBA1
25
*
Foci per cell
20
15
** *
p<0.013
10
5
* : P<0.01
**: P<0.004
G
y
1
G
y
5
0.
0
G
y
0
FACS Analysis
1 hrs post treatment
24 hrs post treatment
Comet Assay
Comet Assay
Control
ZRBA1
ZRBA1 and radiation
Radiation
EGFR Inhibitory Activity (ZRBA1)
EGFR Signaling Pathway
RAF
TGF
MEK
EGFR
HER2
RAS
ERK1/2
P TK
Serine112
PI3K
P
AKT
P
BAD
Serine136
Apoptosis
P
Caspase 9
Caspase 3

Over-expression of EGFR associates
with activation of the AKT pathway.
Survival
Conclusions

Significant cell killing in cells exposed to combination of ionizing
radiation with ZRBA1.

Higher G2M arrest in cells exposed to the combined treatment.

The most effective schedule for this combination is administration of
drug before and / or concurrent with radiation.

ZRBA1 potentiates the effect of radiation in MDA-MB-468 cells.

The combination of IR and ZRBA1 induces the formation of γH2AX foci.
Future Plans

Better understanding of the molecular mechanism induced by this novel
therapeutic approach.

Evaluate the status of DNA repair proteins.

Evaluate the expression levels of antiapoptotic and proapoptotic proteins.

Investigation for other possible modes of cell death.

In-vivo correlative studies.
Cancer Stem Cells

Self-renewing cell

Pluripotent cell: recapitulate
its tumor in SCID mice
CSC: mechanisms of
resistance
CSC: RT resistance

CSC enrichment

Chk1/Chk2 inhibitor
S. Bao Nature 444, 2006
CSC: RT resistance

apoptosis
Cell cycle arrest
CSC: DNA damage repair
kinetics
Future directions

Clinical models for identification of RT resistance



Rectal cancer
CNS tumors
Zevalin resistant lymphoma
Acknowledgments

Dr. Thierry Muanza
- Mitra Heravi
- Lillian Lee
- Azusa Maeda
- Ava Schlisser

Dr. Danuta Radzioch

Dr. Bertrand Jean-Claude (Cancer Drug Research Laboratory)
- Dr. Zakaria Rachid
- Margarita Todorova
Thank You!