Novel Array-Based Target Identification for Synergistic Sensitization of Breast Cancer to Herceptin

Farah Rahmatpanah, Zhenyu Jia, Tatsuya Azum, Eileen Adamson, Ryan Alipio, Becky Pio, Frank Jones, Dan Mercola. Chiponchip analysis of mechanism of action of HER2 inhibition in breast cancer cell linesProceedings of the 100th Annual Meeting of the American Association for Cancer Research; 2009, April 18-22, 2009; Denver, CO, abstract #1030. 6. INVENTIONS, PATENTS, LICENCES. (Nothing to Report) Pending support. (Nothing to report). 7. CONCLUSION. Summarize the results to include the importance and/or implications of the completed research and when necessary, recommend changes on future work to better address the problem. A “so what section” which evaluates the knowledge as a scientific or medical product shall also be included in the conclusion of the report. HER2-positive breast cancer accounts for 25% of all cases and has a poor prognosis. Although progress has been made in understanding signal transduction, little is known of how HER2 achieves gene regulation. We performed whole genome expression analysis on a HER2 + and HER2 breast cancer cell lines and compared these results to expression in 812 primary tumors stratified by their HER2 expression level. Chip-on-chip with anti-RNA polymerase II was compared among breast cancer cell lines to identify genes that are potentially activated by HER2. The expression levels of these HER2-dependent POL II binding genes were determined for the 812 HER2+/breast cancer tissues. Genes differentially expressed between HER2+/cell lines were generally regulated in the same direction as in breast cancer tissues. We identified genes that had POLII binding in HER2 + cell lines, but without significant gene expression. Of 737 such genes “poised” for expression in cell lines, 113 genes were significantly differentially expressed in breast tumors in a HER2-dependent manner. Pathway analysis of these 113 genes revealed that a large group of genes were associated with stem cell and progenitor cell control as indicated by networks centered on NANOG, SOX2, OCT3/4. HER2 directs POL II binding to a large number of genes in breast cancer cells. A “poised” class of genes in HER2 + cell lines with Research Technical Report, D. Mercola, w81xwh-06-1-0253 10 POLII binding and low RNA expression but is differentially expressed in primary tumors, strongly suggests a role of the microenvironment and further suggests a role for stem cells proliferation in HER2-regulated breast cancer tissue. of Herceptin is being developed as an improvement of the identification of gene targets of cisplatin. 8. REPORTABLE OUTCOMES. Dr. R. Rahmatpanah was awarded a training grant fellowship postion of the UCI Cancer Research Institute, May, 2009 for 2 years. 9. OTHER ACHIEVEMENTS. (Nothing to Report) 10. REFERENCES: List all references pertinent to the report using a standard journal format (i.e. format used in Science, Military Medicine, etc.). Shilpi Arora, Yipeng Wang, Zhenyu Jia, Saynur Vardar-Sengul , Ayla Munawar, Kutbuddin S. Doctor, Michael Birrer, Michael McClelland, Eileen Adamson, Dan Mercola. Egr1 regulates the coordinated expression of numerous EGF receptor target genes as identified by ChIP on chip. Genome Biology 2008, 9:R166 [Epub ahead of print]. Jun Hayakawa, Shalu Mittal, Yipeng Wang, Kemal Korkmaz, Mashide Ohmichi, Eileen Adamson, Michael McClelland, Dan Mercola. Identification of genes bound and regulated by ATF2/c-Jun following genotoxic stress. Molecular Cell 2004;16:521-535. Yipeng Wang, Qiuju Yu, Ann Cho, Shalu Mittal, Gaelle Rondeau, Dan Mercola, John Welsh, Michael McClelland, Survey of differentially methylated promoters in prostate cancer cell lines. Neoplasia 2005;8:748-760. Wang, Yipeng, Jun Hayakawa, Fred Long, Qiuju Yu, Ann H Cho, Gaelle Rondeau, John Welsh, Shalu Mittal, Ian de Belle, Eileen Adamson, Michael McClelland, and Dan Mercola. “Promoter Array” studies identify cohorts of genes directly regulated by methylation, copy number change, or transcription factor binding in human cancer cells. Annals of the New York Academy of Sciences, 2005;1058: 162-185. Rahmatpanah, Farahnaz, Zhenyu, Jia, Xin Chen, Jessica E. Char, Benzho Men Anna-Clara Franke , Frank E. Jones, Michael McClelland, Dan Mercola. Class of genes in the HER2 regulon that is poised for transcription in breast cancer cell lines and expressed in human breast tumors. Oncotarget, (in press). 11.APPENDICES: Attach all appendices that contain information that supplements, clarifies or supports the text. Examples include original copies of journal articles, reprints of manuscripts and abstracts, a curriculum vitae, patent applications, study questionnaires, and surveys, etc. Research Technical Report, D. Mercola, w81xwh-06-1-0253 11 Figure 1. Example of the our fabricated promoter array hybridized with DNA from HER2 amplified human breast cancer cells that was purified using antibody to bound POLII mixed to genomic DNA control . The fluorescent signals are appear against a negative background indicating excellent signal to noise characteristics and numerous “spots” are red or green indicating dominance by either the POLII bound DNA or the control showing excellent competitive hybridization with a large dynamic range. Research Technical Report, D. Mercola, w81xwh-06-1-0253 12 Figure 2. An example of hydrization of DNA of human breast cancer cells with amplified HER2 hybridized in competition with genomic DNA to commercial Agilent oligonucleotide arrays. These arrays provide prmoter binding information for over 17,000 human genes compared to 10,525 of our fabricated arrays. We are proceeding with our analysis with both platforms as the overlapping genes will provide a stringent measure of reproducibility. Table 1. Examples of Gene promoters Significantly (p < 0.03; 3 or more probe sets) bound by POLII in high HER2-expressing cells compared control cells. Pathway Pathway Member with gene promoter bound down stream of HER2