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

Normal Breast Tissue

WIT medium

MEGM medium

BPEC Luminal

HMEC Myoepithelial

Transformation hTERT

SV-40 LT/st H-RasV12

BPLER

HMLER Weakly tumorigenic Non metastatic

104 fold more tumorigenicLung metastases

Ince et al., Cancer cell, 2007

Importance of normal cell state in the ability of Myc/p53- combination to transform

hepatoblasts into malignancy

Zender et al., CSH symposia on Quantitative Biology, 2005

P53-/-

P53-/-

Tumor

No Tumor

Myc

Myc

Day 14

Day 18

What change in the 4 days causes such a difference?

Phenotype Comparison

D18/D18

Powers and Chanrion (Unpublished)

D13/D13

Phenotype Comparison D18/D13

Powers and Chanrion (Unpublished)

Method of Ranking Pathways

ScoringMetric

Microarray

Data

Annotations

A score

for each

Pathway: indicates

how much it

was affected

by the condition

Many Different ScoringMetrics Available

PPARARXRA

RXRAPPARG

Adipocyte differentiation

Adaptive thermogenesis

Cell survival

Ubiquitination

Gluconeogenesis

Lipid metabolism (ketogenesis, lipid transport, lipogenesis, cholesterol metabolism, fatty acid oxidation

PPARG Pathway

Knockdown by shRNA

No Tumor

LMS-Myc

D18

P53-/-

Tumor

sh PPAR pathway

P53-/- ?

Activation of PPARG

D13

Tumor

LMS-MycP53-/-

No Tumor

PPAR pathway Activation

P53-/- ?

Shuttle shRNAs from V2 library into LMP vector

16 total LMP-vector shRNA

8 targeting PPARG

2 targeting PPARA

6 targeting RXRA

PurorPPGK GFPIRESLTR + LTR

X SR

5’miR30 3’miR30

B

Unique sites:B = Bgl IIR = EcoR IX = Xho IBs = BstX IS = Sal IBs

PG6

100bp ladderPG8PG7

Ongoing experiments . . .

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

Dr. Scott Powers Dr. Maia Chanrion

C. Bliss Memorial Fund

Cold Spring Harbor Laboratory

Woodbury Genome Center

Powers Lab

Howard Hughes Medical Institute