Alterations in the expression of the genes that control stem cell differentiation and self-renewal often contribute to tumorigenesis. Our laboratory has studied the mechanisms by which retinoids (vitamin A (retinol) and its derivatives and metabolites), control gene expression in both normal stem cells and in tumors. Over this past grant period we have developed evidence that: a) the polycomb group (PcG) protein(s) play a major role in repressing retinoid signaling in embryonic stem (ES) cells;b) these polycomb mediated inhibitory signals are altered in tumor cells;and c) specific retinoid receptors act in concert with transcriptional coactivators in a gene specific context. Our immediate goal in the next grant period is to delineate the mechanisms by which the polycomb group mediated transcriptional repressive pathways and the retinoid transcriptional signaling pathways intersect.
Our Specific Aims for the next grant period: 1) We will elucidate the mechanisms by which the polycomb proteins inhibit retinoid transcriptional activation and retinoic acid induced cell differentiation. This will be accomplished by a combination of ChIP (chromatin immunoprecipitation) assays;overexpression or reduced expression of key transcriptional regulatory proteins such as pCIP (SRC3), EZH2, SUZ12, and JMJD3 in F9 and ES Wt and RAR1, 2, and 3 null cells;and deletion of DNA regulatory elements such as the Hoxa1 RARE, followed by analysis of PcG protein binding. We also propose a comparison of 3T3 cells with H-Ras transformed 3T3 cells to assess how an oncogenic protein perturbs retinoid signaling. 2) We plan to define the domains of the specific retinoic acid receptors (RARs) 1, 2, and 3 (i.e. the receptors for all-trans retinoic acid (RA) and other retinoids) and the coactivators for these receptors that control the transcriptional events which result in cell differentiation (and, thus, aid in inhibiting tumor cell proliferation). 3) Finally, as part of our goal of understanding retinoid regulation of stem cell differentiation, we will undertake a genome-wide search for primary target genes of the RARs through the use of ChIP-on-chip (tiling) arrays or ChIP-seq technology. This approach will allow us to obtain key information about the genes transcriptionally activated as ES cells differentiate along different pathways. For these proposed experiments we will utilize (a) murine ES and teratocarcinoma stem cell lines with both alleles of each of the individual RARs "knocked out" by homologous recombination;(b) ES cell lines with some of the "downstream" RAR target genes "knocked out";and (c) mice that have each of the individual RARs knocked out by homologous recombination. This proposed research will increase our knowledge of the molecular mechanisms by which signaling by retinoids is accomplished, provide new knowledge that will be useful for improvement of 'differentiation therapy'for cancer, delineate the mechanisms by which retinoids signal so that we will understand why some tumors are resistant to retinoid therapy, and allow us to manipulate the expression or function of polycomb repressive proteins to increase the effectiveness of retinoid based therapies and differentiation strategies for stem cells.

Public Health Relevance

Cancer is a dangerous and dreaded disease. This research project will allow us to understand how normal mouse stem cells differentiate or mature in response to vitamin A, a vitamin required for health and without which we would all die. Our new research findings will allow us to improve therapies for cancer treatment, including new therapies in which chemicals related to vitamin A force cancer cells to differentiate and become more like normal cells (differentiation therapy for cancer).

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA043796-23
Application #
8269117
Study Section
Chemo/Dietary Prevention Study Section (CDP)
Program Officer
Jhappan, Chamelli
Project Start
1987-07-15
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
23
Fiscal Year
2012
Total Cost
$338,610
Indirect Cost
$138,249
Name
Weill Medical College of Cornell University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Orfali, Nina; McKenna, Sharon L; Cahill, Mary R et al. (2014) Retinoid receptor signaling and autophagy in acute promyelocytic leukemia. Exp Cell Res 324:1-12
Urvalek, Alison M; Gudas, Lorraine J (2014) Retinoic acid and histone deacetylases regulate epigenetic changes in embryonic stem cells. J Biol Chem 289:19519-30
Urvalek, Alison; Laursen, Kristian Bruun; Gudas, Lorraine J (2014) The roles of retinoic acid and retinoic acid receptors in inducing epigenetic changes. Subcell Biochem 70:129-49
Osei-Sarfo, Kwame; Gudas, Lorraine J (2014) Retinoic acid suppresses the canonical Wnt signaling pathway in embryonic stem cells and activates the noncanonical Wnt signaling pathway. Stem Cells 32:2061-71
Hedblom, Andreas; Laursen, Kristian B; Miftakhova, Regina et al. (2013) CDK1 interacts with RARýý and plays an important role in treatment response of acute myeloid leukemia. Cell Cycle 12:1251-66
Ricard, Megan J; Gudas, Lorraine J (2013) Cytochrome p450 cyp26a1 alters spinal motor neuron subtype identity in differentiating embryonic stem cells. J Biol Chem 288:28801-13
Benoit, Yannick D; Witherspoon, Mavee S; Laursen, Kristian B et al. (2013) Pharmacological inhibition of polycomb repressive complex-2 activity induces apoptosis in human colon cancer stem cells. Exp Cell Res 319:1463-70
Kashyap, Vasundhra; Ahmad, Shafqat; Nilsson, Emeli M et al. (2013) The lysine specific demethylase-1 (LSD1/KDM1A) regulates VEGF-A expression in prostate cancer. Mol Oncol 7:555-66
Laursen, Kristian B; Mongan, Nigel P; Zhuang, Yong et al. (2013) Polycomb recruitment attenuates retinoic acid-induced transcription of the bivalent NR2F1 gene. Nucleic Acids Res 41:6430-43
Kashyap, Vasundhra; Laursen, Kristian B; Brenet, Fabienne et al. (2013) RAR? is essential for retinoic acid induced chromatin remodeling and transcriptional activation in embryonic stem cells. J Cell Sci 126:999-1008

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