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Novel Array-Based Identification of Genes Causing Chemotherapy Resistance by Breast Cancer
Tumor Cell Biology IV
Herceptin (trastuzumab) in combination with platinum salts is a promising new synergistic combination therapy for HER2-positive metastatic breast cancer. This is due in part to Herceptin-induced inhibition of DNA repair which reverses cisplatin-DNA adducts thereby showing the importance of DNA repair. We have discovered a second and independent mechanism that induces rapid DNA repair. Genotoxic stress of cisplatin treatment activates the Jun Kinase (JNK) pathway. Using a high throughput “promoter” array-based analysis we observed that JNK promotes the expression of numerous DNA repair genes which coincides with accelerated DNA repair and resistance of cell killing. JNK acts c-Jun and ATF2 to form an activated AP-1 complex which binds to and induces the transcription of a large cadre of over 269 genes. Chromatin immunoprecipitation (ChIP) of phospho-c-Jun and phospho-ATF2 promoter-complexes was used to isolate promoters that were then identified by hybridization to prototype arrays of 3083 known genes leading to identification of 23 known DNA repair genes. Validation studies confirmed expression at the mRNA and protein level and induction was completely reversed by siRNA to JNK. The results coincide with accelerated DNA repair and decreased cell killing by cisplatin. We propose to exploit the knowledge of these genes and the JNK mediators, c-Jun and ATF2, to develop inhibitors of DNA repair and confirm that inhibition augments Herceptin-cisplatin therapy in vitro . The proposed studies will utilize expanded arrays of 10,012 unique promoter sequences. Aim 1 is the identification of DNA repair genes that are specific to breast cancer cells following cisplatin and Herceptin treatment. We have expanded the cDNA microarray to 10,012 promoters and will employ a proven chip-on-chip strategy to identify the DNA repair genes. We will test that blocking induction of these genes enhances cell killing by cisplatin/Herceptin using siRNA to JNK established in this lab. Aim 2 is to test whether inhibition of JNK-dependent DNA repair is synergistic with Herceptin-cisplatin combination therapy. We will develop new siRNA reagents to inhibiting individual DNA repair genes identified in Aim 1. These new inhibitors will be tested in combination with Herceptin. These studies will determine new combination therapy that is tumor-specific and gene-specific and acts synergistically to achieve enhanced tumor cell killing in combination with Herceptin.
Herceptin is an antibody that is promising to be a major advance in the treatment of advanced breast cancer which is come in patients with tumors that make a protein known as HER2. Indeed when this antibody is used with cisplatin, the results appear better than expected from the sum of each treatment alone. This “super-additive” appears may be due to the fact that these two drugs inhibit distinct pathways Cisplatin damages the genetic material (DNA), which blocks the ability of the tumor cells to multiply. Herceptin binds to a tumor cell surface protein, HER2 and blocks its function. HER2 signals the cells to make proteins that repair DNA which reverses the effect of cisplatin. These results show us the importance of blocking the DNA repair process in over-coming cancer. We have discovered a second way that cancer cells turn on DNA repair. When cancer cells are treated with cisplatin, a “stress” response occurs that causes the cells to produce a large group of 23 DNA repair proteins. We propose to develop inhibitors of the second drug resistance pathway thereby providing a further enhanced therapeutic response to cisplatin/Herceptin therapy. Two aims have been designed to achieve this goal. Aim 1 is to identify the DNA repair genes specially used by breast cancer cells. We have developed a new high throughput genomic-based method for this purpose and published the identification of the 23 genes using this approach. We have expanded this method recently to sample over 10,000 proteins. We will use this method to identify breast cancer specific genotoxic stress-induced DNA repair proteins. Aim 2 is to develop blockers of the various DNA repair proteins that we have found in Aim 1. A new and powerful method that we have used to make inhibitors that block the ability of cells to make a particular protein is to use “siRNA” or small interfering RNA. We will use this method to block the ability of tumor cells to use each DNA repair protein and determine which protein is crucial for resistance to therapy. We will test that normal cells are not significantly impacted. The new inhibitor will be combined with Herceptin to test that the combination provides a super-additive tumor cell killing result. Once this is demonstrated, we can conclude that the new method of blocking DNA repair is a major potential advance for the use of Herceptin-cisplatin therapy.