Radiotherapy & Resistance

2E Conférence Québécoise Sur La Résistance Thérapeutique du CancerQ-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

ATM

G1 arrest

RepairG

2 ar

rest G1 arrest

Apoptosis

G2 arrest

pH2XA

P53

BAX

Cytochrome c

Apaf1 Caspase 9

Caspase 6Caspase 3

Apoptosis

P53

Cycline B

N Cycline-B-p34 cdc2

p21 WAF1 / CIPIcdK /Cycline D

Cycline E

pRb

E2F

pRb

E2F

Smac/ Diablo

Survivin

DNA Repair Mechanisms

ATM DSB

Rad51 /Rad52

Rad52/MRE11/NBSI

BRAC1/BRAC2

Completion of DSB repair

DNA-PKcs / Ku80/70

MRE11/Rad51/NBSI

DNA ligase IV, XRCC4

RNA Pol II or XPC

XPD / XPB

ERCC1 / XPG

Completion of repair

DNA Pol / ligase

DNA Pol / DNA ligase III

XRCC1

BER NHEJ

HR

pH2aXNER

SSB

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 DNA Cycling tissues

Hypoxic 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

0

10

20

30

40

50

60

70

80

90

100

110

0 10 20 30 40 50 60 70 80 90 100 110

Drug Concentration (uM)

Per

cent

Con

trol

Cel

l Via

bilit

y

ZRBA1 with Radiation

Iressa with Radiation

ZRBA1

Iressa

Heravi, Muanza et al. ACD 20, 2009

Cell Proliferation Assay (MTT)

0

20

40

60

80

100

120

140

No XRT RX then XRT RX+XRT XRT then RX

Per

cent

cel

l via

bilit

y

Control

ZRBA1

**

**

p<0.0049

**: P<0.01

Colony Forming Assay

0 2 4 6 8 100.0001

0.001

0.01

0.1

1ZRBA1

Iressa

Control

Radiation Dose (Gy)

Sur

viva

l Fra

ctio

n

Radiation Dose (Gy) DER

2 4 6

1.62.22.9

γH2AX Immunofluorescent Staining

Control

1 Gy

0.5 Gy

γH2AX Immunofluorescent Staining

Control

ZRBA1 18uM

ZRBA1 9uM

γH2AX Immunofluorescent Staining

Control 0.5 Gy

ZRBA1 9uM 0.5 Gy and ZRBA1 9uM

γH2AX Immunofluorescent Staining

Control 1 Gy

ZRBA1 9uM 1 Gy and ZRBA1 9uM

γH2AX Immunofluorescent Staining

0 Gy

0.5

Gy1

Gy0

5

10

15

20

25

Control

9 uMZRBA1

18 uM ZRBA1

*** *

* : P<0.01**: P<0.004

p<0.013

Fo

ci p

er

cell

FACS Analysis

1 hrs post treatment

24 hrs post treatment

Comet Assay

Comet Assay

Control ZRBA1

Radiation

ZRBA1 and radiation

EGFR Inhibitory Activity (ZRBA1)

EGFR Signaling Pathway

TK

TGF

RAS

RAF

ERK1/2

BAD

ApoptosisCaspase 9

Caspase 3

Survival

AKT

EGFR

PSerine136

Serine112

PPI3K

P

Over-expression of EGFR associates

with activation of the AKT pathway.

P

HER2

MEK

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!