The goal of this project renewal is to establish the biological rationale and potential clinical significance of selectively targeting the HER2 (c- erbB-2/neu) promoter to repress its transcription in breast cancer cells that overexpress the receptor product of this oncogene. Agents designed to downregulate overexpressed p185HER2 growth factor receptor are showing promise in preclinical and clinical studies. However, efforts supported by this grant have revealed mechanisms of resistance to HER2 receptor- targeted therapy and point to a need for alternative strategies to inactivate this critical oncogene/receptor system by specifically targeting its transcription machinery. Addressing this need, the project's most recent accomplishments include evidence that: i) one of four ETS family transcription factors (GABPalpha, elk-1, elf-1, and PEA3) expressed in human breast cancer cells accounts of HER2 overexpression by binding to a consensus ets element (GAGGAA) critical to HER2 promoter function, and ii) triplex-forming oligonucleotide sequences that specifically recognize and ind the polypurine tract adjacent to the ets element can repress the formation of nascent HER2 transcripts. This project will first identify key components of the HER2 promoter-binding ETS complex and establish the critical nature of the ets response element relative to other upstream response elements, as rationale for two proposed strategies to inhibit HER2 transcription. These strategies will then be compared for their ability to downregulate HER2 receptor expression, reduce tumor cell growth and alter the phenotype of HER2- positive breast cancer cells. The first strategy will employ a new oligonucleotide-producing vector that generates large quantities of sequence-specific anti-HER2 Triplex-forming RNA Olignucleotides (TRO) within the nuclei cultured breast cancer cells. In vitro testing of this vector is complete and transfection studies have begun to assess the ability of intracellulary generated TRO to bind multiple copies of the HER2 promoter and specifically repress HER2 transcription. The second strategy involves construction of an ETS-specific fusion vector designed to produce a nuclear-localizing protein chimera composed of two fused modular domains, an 85 amino acid ETS DNA-binding domain (recognizing only the GAGGAA element in the HER2 promoter) and a recently described potent transcription repressing domain. Construction of a prototype form of this vector has begun and when HER2 promoter-binding and intracellular accumulation of the ETS protein chimera are optimized, the ability of the chimeric protein to inhibit HER2 transcription will be compared with that of the anti-HER2 TRO in the same HER2-positive breast cancer cell lines. Initially, the anti-HER2 TRO and ETS chimera will be assessed by cell- free assays as well as transient transfection into HER2-positive breast cancer cells to measure their ability to inhibit transcription from co- transfected or endogenously amplified HER2 promoters. Later, stable transfectants will be created bearing the ectopically introduced anti- HER2 genes encoding either ETS chimera or TRO in both HER2-positive and HER2-negative breast cancer cells. These breast cancer sublines overexpressing either anti-HER2 TRO or the ETS chimera will be compared for reduction of HER2 mRNA and receptor levels, alterations in tumor cell phenotype and expression of other ETS-regulated genes, and changes in tumor cell proliferative capacity and tumorigenicity.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA036773-16
Application #
2747697
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Program Officer
Forry, Suzanne L
Project Start
1983-08-15
Project End
2002-11-30
Budget Start
1999-01-01
Budget End
1999-12-31
Support Year
16
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Marx, Corina; Held, Jason M; Gibson, Bradford W et al. (2010) ErbB2 trafficking and degradation associated with K48 and K63 polyubiquitination. Cancer Res 70:3709-17
Scott, Gary K; Marx, Corina; Berger, Crystal E et al. (2008) Destabilization of ERBB2 transcripts by targeting 3'untranslated region messenger RNA associated HuR and histone deacetylase-6. Mol Cancer Res 6:1250-8
Bhaumik, D; Scott, G K; Schokrpur, S et al. (2008) Expression of microRNA-146 suppresses NF-kappaB activity with reduction of metastatic potential in breast cancer cells. Oncogene 27:5643-7
Zhou, Yamei; Yau, Christina; Gray, Joe W et al. (2007) Enhanced NF kappa B and AP-1 transcriptional activity associated with antiestrogen resistant breast cancer. BMC Cancer 7:59
Goga, Andrei; Benz, Christopher (2007) Anti-oncomir suppression of tumor phenotypes. Mol Interv 7:199-202, 180
Marx, Corina; Yau, Christina; Banwait, Surita et al. (2007) Proteasome-regulated ERBB2 and estrogen receptor pathways in breast cancer. Mol Pharmacol 71:1525-34
Scott, Gary K; Goga, Andrei; Bhaumik, Dipa et al. (2007) Coordinate suppression of ERBB2 and ERBB3 by enforced expression of micro-RNA miR-125a or miR-125b. J Biol Chem 282:1479-86
Neve, Richard M; Parmar, Hema; Amend, Cliff et al. (2006) Identification of an epithelial-specific enhancer regulating ESX expression. Gene 367:118-25
Mattie, Michael D; Benz, Christopher C; Bowers, Jessica et al. (2006) Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies. Mol Cancer 5:24
Benz, Christopher C; Atsriku, Christian; Yau, Christina et al. (2006) Novel pathways associated with quinone-induced stress in breast cancer cells. Drug Metab Rev 38:601-13

Showing the most recent 10 out of 55 publications