Summer Undergraduate Research Program - Fred Hutchinson ...

Investigating a Small Molecule Inhibitor of Aurora
Kinase-A in Ras-driven Pancreas Cancer Cells
Brian W. Wakefield1,2 ; Amy E. Chang2 ; Vikas K. Goel, Ph.D2 ; Sunil R. Hingorani, M.D., Ph.D. 2
1Whitman College, Walla Walla, WA; 2Fred Hutchinson Cancer Research Center, Seattle, WA
2010 Best Poster Presentation Award
RalGTPase Pulldown Assay: RalA activity decreases with AKAi-1.
Results
Cell Proliferation: AKAi-1 inhibits cell growth in KPC cells.
AKA
P + P+
Fig. 10: Western Blot. KPC cells
treated with AKAi-1 or vehicle for 36
hours. Signal intensity normalized to
GST loading control. Inhibition of
AKA by AKAi-1 causes downregulation
of active RalA.
P + P+
AKA
Conclusions
AKAi-1 treatment in primary KPC cell lines:
Inhibits cell proliferation with increased G2/M arrest
Increases apoptosis
These effects occurred in a concentration-dependent manner.
Abstract
Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancerrelated
deaths in the United States and, with a five-year survival rate of less
than 5%, is almost uniformly fatal. Current therapies are only able to improve
survival by several weeks and new approaches to the study of this disease are
therefore imperative. Over 90% of pancreas cancers have an activating
mutation in the KRAS proto-oncogene while 75% have an inactivating
mutation in the tumor suppressor gene, TP53. We have shown that
endogenous expression of KrasG12D and Trp53R172H in the pancreata of mice
induces disease that mimics human pancreas cancer from precursor lesions to
fully invasive and metastatic PDA. Direct inhibition of oncogenic KRAS in
patients has proved challenging, causing a shift in focus toward its downstream
effectors. The small GTPase, RalA, is downstream target of Ras signaling,
necessary for Ras-mediated oncogenesis, and is also activated by Aurora
Kinase A (AKA). We describe the effects of a small molecule inhibitor of
AKA (AKAi-1) on primary tumor cells derived from the pancreata of mice
expressing oncogenic KrasG12D and mutant Trp53R172H . Standard assay
methods for apoptosis, proliferation, and cell cycle progression were used.
These experiments were designed to determine whether this drug should
proceed to preclinical trials in the autochthonous mouse model.
CDC25
No additive or synergistic effects were observed with simultaneous treatment
of AKAi-1 and Gemcitabine.
Fig. 3: Aurora Kinase A and Kras converge to activate the small GTPase RalA
These data suggest that AKAi-1 may be effective in treating Ras-driven
carcinomas and justify testing this inhibitor in our preclinical mouse model.
AKAi-1
1 2 5 10 - - - 1 2 5 10 1 2 5 10
(µM)
GEM
- - - - 1 5 10 1 1 1 1 5 5 5 5
(nM)
Fig. 5: Growth inhibition by AKAi-1 in KPC cells. Concentration-dependent inhibition was seen in
cells treated with AKAi-1 as compared to DMSO control. No additive or synergistic effects were
observed from co-administration of AKAi-1 with Gemcitabine.
Cell Cycle: AKAi-1 promotes G2/M arrest in KPC cells
Vehicle
Cell Cycle
Progression
Background
Targeted expression of oncogenic KrasG12D to the pancreas initiates
pancreas cancer1 Concomitant expression of mutant Trp53 cooperates to accelerate widely
metastatic disease2 Future Directions
157
To gain a better understanding of the molecular interactions between Kras
and RalA, we will quantify the effects on RalA activity of silencing KrasG12D using shRNA in an inducible system.
Mouse PDA Cells NIH3T3 Cells
Control V33 V28 V24 V14 C. V28 V24 V14
DOX - + - + - + - + - + + + + +
AKAi-1
0.5 µM
AKAi-1
2.5 µM
AKAi-1
- 0.5 1 2.5 5 - - - 0.5 0.5 1 1
(µM)
GEM
- - - - - - 2 5 2 5 2 5
(nM)
Fig. 6: Effect of AKAi-1on cell cycle progression in KPC cells. Concentration-dependent increase in
G2/M arrest was seen in AKAi-1-treated cells. No additive or synergistic effects were observed from coadministration
of AKAi-1 with Gemcitabine. Fig7: Representative plots.
Apoptosis: AKAi-1 increases apoptosis in KPC cells
Materials and Methods
Experiments were conducted in primary cell lines derived from murine
pancreatic tumors (KPC):
KPC
LSL-KrasG12D activating mutation
LSL-Trp53R172H mutation
Fig. 1: Conditional LSL-KRASG12D and LSL-Trp53R172H (KPC) alleles and generation
of functional mutant via Cre recombinase2 Pdx-1-Cre
Fig. 11: Western blot. KPC cells (left) infected with a Doxycyclin-inducible TripZ vector (V) carrying
KrasG12D shRNA show silencing of oncogenic Kras when exposed to Doxycyclin. NIH3T3 cells (right)
Fig. 2b: Specimen
from same mouse
showing hepatic
metastases (black
arrows) and a large
head-of-the-pancreas
primary tumor ( * ) 2
Fig. 2a: Gross
pathological
photograph of
metastatic
PDA in a KPC
mouse2 expressing wild-type Kras show no effect.
Ras activity will be quantified with a RasGTPase pull-down assay
RalA activity will be quantified with a RalGTPase pull-down assay
We will administer AKAi-1 to KPC animals with established tumors alone
and in combination with Gemcitabine. The results of the in vivo study together
with the in vitro data will help determine whether this drug should proceed to
clinical trials.
Vehicle Control
AKAi-1 0.5µM AKAi-1 1.0µM
Fig. 8: Representative apoptosis flow cytometry data from KPC cells. Double positive (upper right,
circled) population represents late apoptotic and dead cells. Annexin V positive population (lower
right) represents early apoptotic cells.
Fig. 4: Western blot. KPC tumor cells show high levels of AKA expression and expression of
RalA
Fig. 2d: Welldifferentiated
liver
metastasis (m) and
surrounding normal
tissue (h) 2
Fig. 2c: Welldifferentiated,
glandular
PDA2 Cell Proliferation: cells were plated in 96-well plates and exposed to a
spectrum of AKAi-1 and Gemcitabine concentrations alone and in
combination. Proliferation was analyzed by WST-1 and absorbance was
read at 450nm.
References
1.Hingorani, Sunil et al. “Preinvasive and Invasive Ductal Pancreatic Cancer and its Early Detection in the Mouse.”
Cancer Cell 4(2003): 437-450.
2.Hingorani, Sunil et al. “Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely
metastatic pancreatic ductal adenocarcinoma in mice.” Cancer Cell 7(2005): 469-483
3.Brunner, Thomas et al. “Farnesyltransferase Inhibitors: An Overview of the Results of Preclinical and Clinical
Investigations.” Cancer Research 63(2003): 5656-5668.
4.Lim, Kian-Huat et al. “Aurora-A Phosphorylates, Activates, and Relocalizes the Small GTPase RalA.” Molecular
and Cellular Biology 30(2010): 508-523
Cell Cycle and Apoptosis: cells were plated in 60mm plates and
exposed to a spectrum of AKAi-1 and Gemcitabine concentrations alone and
in combination. Cell cycle was analyzed by propidium iodide staining and
flow cytometry. Apoptosis was analyzed by PE-Annexin V staining and
flow cytometry.
Direct inhibition of constitutively active oncogenic Kras or its upstream
regulators ineffective or impossible in clinical populations 3
Targeted inhibition of downstream effectors of Kras may reveal new
potential therapeutic targets
RalA Activity: Active, phosphorylated RalA was quantified using a
RalGTP assay (MilliporeTM ). Active RalA was pulled down by RalBP1 as
per manufacturer’s instructions and quantified by western blot.
Acknowledgements
Hypotheses
Targeting specific downstream effectors of Kras maybe an effective method
of inhibiting Ras-driven PDA
This program was supported by a research grant from the Howard Hughes Medical Institute and Whitman College.
I would personally like to thank Dr. Sunil Hingorani, Dr. Vikas Goel, Amy Chang, Natalie Wong, Layken Fletcher, Dr.
Paolo Provenzano, and Dr. Libing Feng for their help and guidance during this internship.
AKAi-1
- 0.5 1 2.5 5 - - - 0.5 0.5 1 1
(µM)
GEM
- - - - - - 5 10 5 10 5 10
(nM)
Fig. 9: Effect of AKAi-1on apoptosis in KPC cells. Concentration-dependent increase in apoptosis was
seen in AKAi-1-treated cells. Effect plateaued near IC50 value for AKAi-1 (2.06 µM). Concentrationdependent
increase in apoptosis was also observed for Gemcitabine treatments. No additive or
synergistic effects were observed from co-administration of AKAi-1 with Gemcitabine.
Knockdown of KrasG12D by shRNA: Cells were infected with
Doxycyclin-inducible TripZ vectors containing Kras shRNA. Knockdown
efficiency was estimated by western blot.
Using RalA to link Aurora Kinase A (AKA) and Kras validates AKA as a
therapeutic target in treatment of PDA