Influence of initial differentiated state of the normal
cell on the final tumorigenic phenotype
Yesenia Correa
Undergraduate Research Program
Cold Spring Harbor Laboratory
Dr. Maia Chanrion
Dr. Scott Powers
Influence of normal cell on neoplastic phenotype
HMEC
HMLER
Myoepithelial
Weakly tumorigenic
Non metastatic
MEGM medium
Transformation
Normal Breast
Tissue
hTERT
SV-40 LT/st
H-RasV12
WIT medium
BPEC
Luminal
BPLER
104 fold more tumorigenic
Lung metastases
Ince et al., Cancer cell, 2007
Importance of normal cell state in the ability of
Myc/p53- combination to transform
hepatoblasts into malignancy
Day 14
P53-/-
Myc
Tumor
Day 18
P53-/-
Myc
No Tumor
What change in the 4 days causes such a difference?
Zender et al., CSH symposia on Quantitative Biology, 2005
Phenotype Comparison
D13/D13
D18/D18
Powers and Chanrion (Unpublished)
Phenotype Comparison D18/D13
Powers and Chanrion (Unpublished)
Method of Ranking
Pathways
Microarray
Data
Scoring
Metric
A score
for each
Pathway:
indicates
how much it
was affected
by the condition
Annotations
Many Different Scoring
Metrics Available
PPARG Pathway
Lipid metabolism
(ketogenesis, lipid transport, lipogenesis, cholesterol
metabolism, fatty acid oxidation
PPARA
RXRA
Adipocyte differentiation
Adaptive thermogenesis
PPARG
RXRA
Cell survival
Ubiquitination
Gluconeogenesis
Knockdown by shRNA
D18
No Tumor
P53-/LMS-Myc
Tumor
P53-/-
?
sh PPAR pathway
Activation of PPARG
D13
Tumor
P53-/LMS-Myc
No Tumor
P53-/-
?
PPAR pathway Activation
Shuttle shRNAs from V2 library
into LMP vector
LTR
+
5’miR30
B
3’miR30
PPGK Puror IRES
XR
GFP
Bs
16 total LMP-vector shRNA
8 targeting PPARG
2 targeting PPARA
6 targeting RXRA
Ongoing experiments . . .
LTR
S
PG6
PG7
Unique sites:
B = Bgl II
R = EcoR I
X = Xho I
Bs = BstX I
S = Sal I
PG8
100bp
ladder
Potential cancer therapy: Activation of
the PPARG Pathway using Troglitazone
• Explored as a potential
cancer therapeutic in
animal models and
clinical trials
• Some but not all
studies implicate its
potential therapeutic
utility in liver cancer
Yu et al., Hepatology, 2006
Clonogenic Assay with Troglitazone
• Mouse Cell lines:
– Day 14: 18-8/1 and 17-8/3
• Tumor cell line from D14 p53-/- and myc hepatoblasts
– Day 18: IM+DLCs, 122-2ve, and 118-1
• Tumor cell line from D18 p53-/-, myc and shRNA
hepatoblasts
Ongoing experiments
• Validation of PPARA, PPARG, and RXRA
shRNA through Western Blot
• Biological validation (tumorigenicity
assays)
• Determinant of troglitazone response?
Acknowledgements
C. Bliss Memorial Fund
Cold Spring Harbor Laboratory
Dr. Scott Powers
Dr. Maia Chanrion
Woodbury Genome Center
Howard Hughes Medical Institute
Powers Lab