Overexpression of PDGFR-beta is an important factor in pancreatic cancer as well as other inflammatory diseases, including arteriosclerosis and therefore is a highly significant molecular target. The long-range goal of this proposal is to develop small molecule therapeutics that will specifically suppress PDGFR-beta gene expression. Our strategy takes advantage of the recent insight into the importance of G-quadruplexes in transcriptional silencing of a number of genes, including c-Myc. Preliminary data reported in this proposal demonstrate that a cluster of four overlapping G-quadruplexes are key elements in the control of PDGFR-beta transcription. We have also demonstrated that known G-quadruplex-interactive compounds have differential effects on PDGFR-beta gene expression dependent on the selectivity for the constituent G-quadruplexes. Transcriptionally induced superhelicity has been demonstrated to be important in the conversion of duplex DNA to G-quadruplex in promoter region. Thus our hypothesis to be tested is that the negative superhelicity induced by transcription provides a real-time feedback mechanism into the NHE in the PDGFR-beta promoter to modulate both the firing rate (cruise control) and activation or silencing (on/off switch) of PDGFR-beta transcription.
The specific aims are: (1) To determine the biological function of the 54-end, mid-54, mid-34, and 34-end G-quadruplex-forming sequences by deletion and mutational analysis of the PDGFR-beta promoter element and then determine the effect of supercoiling on the pattern of G-quadruplex formation in the PDGFR-beta promoter. (2) To determine by NMR the folding patterns and structures of the G-quadruplexes in the PDGFR-beta promoter. (3) To identify specific proteins and small molecules that bind differentially to the constituent G-quadruplexes found in the PDGFR-beta promoter.
For specific aim 1, we will construct supercoiled plasmids containing PDGFR-beta promoter inserts and a luciferase reporter system.
For specific aim 2, high-field NMR will be used to determine the structures of the constituent G-quadruplexes located in the PDGFR-beta promoter element.
For specific aim 3, we will use affinity chromatography and small molecule screening methods to identify proteins and drug-like molecules that bind to the individual G-quadruplexes. The biological effects of these entities will be determined in specific aim 1.

Public Health Relevance

An effective means to turn on and off genes associated with diseases, such as cancer and arteriosclerosis, with small drug-like molecules is still not available. In this project, we take advantage of a new mechanism to achieve this objective. The target is a gene that is commonly involved in pancreatic cancer and arteriosclerosis.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA153821-03
Application #
8257544
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Misra, Raj N
Project Start
2010-07-01
Project End
2015-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
3
Fiscal Year
2012
Total Cost
$351,112
Indirect Cost
$115,999
Name
University of Arizona
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
806345617
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Kendrick, Samantha; Kang, Hyun-Jin; Alam, Mohammad P et al. (2014) The dynamic character of the BCL2 promoter i-motif provides a mechanism for modulation of gene expression by compounds that bind selectively to the alternative DNA hairpin structure. J Am Chem Soc 136:4161-71
Kang, Hyun-Jin; Kendrick, Samantha; Hecht, Sidney M et al. (2014) The transcriptional complex between the BCL2 i-motif and hnRNP LL is a molecular switch for control of gene expression that can be modulated by small molecules. J Am Chem Soc 136:4172-85
Brown, Robert V; Hurley, Laurence H (2011) DNA acting like RNA. Biochem Soc Trans 39:635-40
Brooks, Tracy A; Kendrick, Samantha; Hurley, Laurence (2010) Making sense of G-quadruplex and i-motif functions in oncogene promoters. FEBS J 277:3459-69