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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. 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

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  1. 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

  2. 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

  3. 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

  4. Phenotype Comparison D13/D13 D18/D18 Powers and Chanrion (Unpublished)

  5. Phenotype Comparison D18/D13 Powers and Chanrion (Unpublished)

  6. 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

  7. 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

  8. Tumor ? P53-/- sh PPAR pathway Knockdown by shRNA D18 No Tumor P53-/- LMS-Myc

  9. No Tumor ? P53-/- PPAR pathway Activation Activation of PPARG D13 Tumor P53-/- LMS-Myc

  10. 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 . . .

  11. 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

  12. 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

  13. Ongoing experiments • Validation of PPARA, PPARG, and RXRA shRNA through Western Blot • Biological validation (tumorigenicity assays) • Determinant of troglitazone response?

  14. Acknowledgements C. Bliss Memorial Fund Cold Spring Harbor Laboratory Dr. Scott Powers Dr. Maia Chanrion Woodbury Genome Center Howard Hughes Medical Institute Powers Lab

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