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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. HMEC Myoepithelial. HMLER . Weakly tumorigenic Non metastatic. MEGM medium.

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influence of initial differentiated state of the normal cell on the final tumorigenic phenotype

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

Myoepithelial

HMLER

Weakly tumorigenic Non metastatic

MEGM medium

Transformation

hTERT

SV-40 LT/st

H-RasV12

Normal Breast Tissue

WIT medium

104 fold more tumorigenic

Lung metastases

BPEC

Luminal

BPLER

Influence of normal cell on neoplastic phenotype

Ince et al., Cancer cell, 2007

slide3
Importance of normal cell state in the ability of Myc/p53- combination to transform hepatoblasts into malignancy

Day 14

P53-/-

Tumor

Myc

Day 18

No Tumor

P53-/-

Myc

What change in the 4 days causes such a difference?

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

phenotype comparison
Phenotype Comparison

D13/D13

D18/D18

Powers and Chanrion (Unpublished)

phenotype comparison d18 d13
Phenotype Comparison D18/D13

Powers and Chanrion (Unpublished)

method of ranking pathways
Method of Ranking Pathways

A score

for each

Pathway:

indicates

how much it

was affected

by the condition

Microarray

Data

Scoring

Metric

Annotations

Many Different Scoring

Metrics Available

pparg pathway

PPARA

RXRA

PPARG

RXRA

PPARG Pathway

Lipid metabolism

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

Adipocyte differentiation

Adaptive thermogenesis

Cell survival

Ubiquitination

Gluconeogenesis

knockdown by shrna

Tumor

?

P53-/-

sh PPAR pathway

Knockdown by shRNA

D18

No Tumor

P53-/-

LMS-Myc

activation of pparg

No Tumor

?

P53-/-

PPAR pathway Activation

Activation of PPARG

D13

Tumor

P53-/-

LMS-Myc

shuttle shrnas from v2 library into lmp vector

100bp ladder

PG6

PG7

PG8

Shuttle shRNAs from V2 library into LMP vector

Unique sites:

B = Bgl II

R = EcoR I

X = Xho I

Bs = BstX I

S = Sal I

16 total LMP-vector shRNA

8 targeting PPARG

2 targeting PPARA

6 targeting RXRA

PPGK

Puror

IRES

GFP

LTR

+

LTR

5’miR30

3’miR30

Bs

S

B

X

R

Ongoing experiments . . .

potential cancer therapy activation of the pparg pathway using troglitazone
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
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
Ongoing experiments
  • Validation of PPARA, PPARG, and RXRA shRNA through Western Blot
  • Biological validation (tumorigenicity assays)
  • Determinant of troglitazone response?
acknowledgements
Acknowledgements

C. Bliss Memorial Fund

Cold Spring Harbor Laboratory

Dr. Scott Powers

Dr. Maia Chanrion

Woodbury Genome Center

Howard Hughes Medical Institute

Powers Lab