This proposal is focused on the molecular mechanisms underlying transcriptional silencing of the platelet-derived growth factor (PDGF) A-chain gene. Polypeptide growth factors (GFs) such as PDGF, EGF and TGFalpha, among many others, are important positive growth effectors in malignant cells, while others such as TGFbeta have been implicated as inhibitory regulators. However, very little is known about the mechanisms through which GF genes are repressed, nor about the mechanisms through which control of GF gene transcription is subverted in cancer. We have identified multiple silencer elements in the 5'-flanking region of the A-chain gene, one of which was localized to a 33 bp sequence and denoted the 5'-S1 nuclease-hypersensitive (5'SHS) silencer. Two 5'SHS-binding factors were identified as NM23-H1 and NM23-H2 (H1 and H2), proteins implicated previously in suppression of metastasis in breast cancer and melanoma. We have also shown that H1 and H2 expression is required for A-chain silencing, and that DNA-binding is required for their silencing and growth-suppressing activities. We have also observed binding of a number of other protein species (p97, p87, p70, p44/48) to the 5'SHS silencer element. These findings appear to place this project in an excellent position to study molecular mechanisms of transcriptional silencing in a GF gene model. The linkage between NM23s and silencer function also provides a foundation for study of mechanisms through which NM23s mediate transcriptional repression and thereby suppress tumor progression. The proposed studies will test the hypothesis that binding of NM23 and other 5'SHS-binding proteins is critical to function of the structurally related 5'SHS and intSHS silencers of the A-chain gene. In addition, we will determine the extent to which DNA-binding and structure-modifying activities of H1 and H2 are required for silencing and growth-suppression. DNA structure will be assessed by nuclease- and chemical-hypersensitivity assays, while DNA-protein interactions will be characterized by electrophoretic mobility shift assay (EMSA), Southwestern blot and DNA footprinting. Silencer function will be determined by transient transfection analysis in a panel of tumor cell lines that exhibit a range of A-chain silencer activities and expression levels of H1 and H2. Together, the proposed studies should provide important new insights into the mechanisms through which precise control of GF gene transcription is compromised in cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA083237-04
Application #
6514195
Study Section
Metabolic Pathology Study Section (MEP)
Program Officer
Perry, Mary Ellen
Project Start
1999-08-01
Project End
2004-05-31
Budget Start
2002-06-01
Budget End
2003-05-31
Support Year
4
Fiscal Year
2002
Total Cost
$213,018
Indirect Cost
Name
University of Kentucky
Department
Pharmacology
Type
Schools of Medicine
DUNS #
832127323
City
Lexington
State
KY
Country
United States
Zip Code
40506
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Leonard, M Kathryn; McCorkle, Joseph R; Snyder, Devin E et al. (2018) Identification of a gene expression signature associated with the metastasis suppressor function of NME1: prognostic value in human melanoma. Lab Invest 98:327-338
Leonard, M Kathryn; Pamidimukkala, Nidhi; Puts, Gemma S et al. (2017) The HGF/SF Mouse Model of UV-Induced Melanoma as an In Vivo Sensor for Metastasis-Regulating Gene. Int J Mol Sci 18:
Novak, Marián; Leonard, Mary Kathryn; Yang, Xiuwei H et al. (2015) Metastasis suppressor NME1 regulates melanoma cell morphology, self-adhesion and motility via induction of fibronectin expression. Exp Dermatol 24:455-61
Kaetzel, David M; Leonard, Mary K; Cook, Gemma S et al. (2015) Dual functions of NME1 in suppression of cell motility and enhancement of genomic stability in melanoma. Naunyn Schmiedebergs Arch Pharmacol 388:199-206
McCorkle, Joseph R; Leonard, Mary K; Kraner, Susan D et al. (2014) The metastasis suppressor NME1 regulates expression of genes linked to metastasis and patient outcome in melanoma and breast carcinoma. Cancer Genomics Proteomics 11:175-94
Jarrett, Stuart G; Novak, Marian; Harris, Nathan et al. (2013) NM23 deficiency promotes metastasis in a UV radiation-induced mouse model of human melanoma. Clin Exp Metastasis 30:25-36
Jarrett, Stuart G; Novak, Marian; Dabernat, Sandrine et al. (2012) Metastasis suppressor NM23-H1 promotes repair of UV-induced DNA damage and suppresses UV-induced melanomagenesis. Cancer Res 72:133-43
Ganguly, S S; Fiore, L S; Sims, J T et al. (2012) c-Abl and Arg are activated in human primary melanomas, promote melanoma cell invasion via distinct pathways, and drive metastatic progression. Oncogene 31:1804-16
Zhang, Qingbei; McCorkle, Joseph R; Novak, Marian et al. (2011) Metastasis suppressor function of NM23-H1 requires its 3'-5' exonuclease activity. Int J Cancer 128:40-50

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