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    Research Grants Awarded

    Gene-Signature Targeting As A Novel Approach For Treating Breast Cancer

    Grant Mechanism:
    Postdoctoral Fellowships

    Scientific Abstract:
    Genetic and Chemical Screens for Suppressors of Oct4 Expression in Breast Cancer BACKGROUND: The fundamental problem of breast cancer treatment is the recurrence of drug-resistant cancer locally, regionally, or distantly. A better understanding of the underlying biology of recurrence has been recently made. Only a small population of breast cancer cells with stem-like properties (cancer stem-like cells) is found to be tumorigenic. Due to their unique properties, cancer stem-like cells are resistant to traditional therapies and responsible for the recurrence. Therefore, the key to curing breast cancer is to target these cells. Oct4 is an embryonic stem cell marker, which keeps cells in a stem-like status and prevents differentiation. It is normally not expressed in somatic cells. However, re-expression of Oct4 is found in breast cancer stem-like cells. Moreovre, the oncogenic role of Oct4 expression is closely linked to the increased activity of stem/progenitor cells and prevention of cells from differentiation, similar to its function in embryonic stem cells. Therefore abolishing the expression of Oct4 is of great importance to target cancer stem like-cells. However, it remains unknown what are intrinsic and extrinsic suppressors of Oct4 expression. This is a critical question to understand how tumorigenic breast cancer cells acquire stem-like properties and to develop effective therapeutic strategy to target these cells. HYPOTHESIS/SPECIFIC AIMS: Suppression of Oct4 expression in breast cancer cells will provide a novel and effective strategy for targeting breast cancer stem-like cells. This proposal is centered to: (Aim1) systematically elucidate intrinsic transcriptional suppressors of Oct4 expression through a functional genetic screen utilizing an enhanced retrovial mutagenesis (ERM) system; and (Aim2) identify drugs that turn off Oct4 expression and/or specifically target Oct4 positive breast cancer cells by utilizing a high-throughput, imaging-based assay system to screen chemical libraries that contains a large collection of candidate compounds and FDA- approved drugs. STUDY DESIGN: Human embryonic stem cells (hESCs) and breast cancer cell line MCF-7 will be stably transfected with GFP reporter driven by a full length human Oct4 promoter region to create hESCs-GFP and MCF-7-GFP cells. hESCs-GFP cells will be infected with the ERM library to insertionally activate the entire genome. Overexpression of Oct4 suppressors by ERM will turn off Oct4 promoter driven GFP signal, which will be identified by FACS. Candidate genes identified via sequencing of inserted ERM tag will be further validated by ectopic expression or siRNA knockdown methods. Previous studies and our observations show that MCF-7-GFP cells contain a heterogeneous population consisting of Oct4 expressing cells (GFP-positive cells) and more differentiated cells without Oct4 expression (GFP-negative cells). The cytotoxic effect will be detected by cell membrane permeability dye labeled with red fluorescence. After exposure to the chemical libraries, effects of drugs on Oct4 expression and survival of Oct4 positive cells will be analyzed based on combinatory responses of two fluorescent signals captured by IN Cell Analyzer 1000. Candidate drugs will be validated by their effects on Oct4 expression and the tumorigenic capacity of cancer stem-like cells. IMPACT: This study will better our understanding of how breast cancer cells acquire stem like properties through the loss of silencing of stem cell genes, which are linked to novel oncogenic pathways. Oct4 is not expressed in normal cells and its re-expression in breast cancer stem-like cells is believed to be the underlying mechanism for cancer recurrence. Therefore, targeting Oct4 will provide a novel and effective therapeutic strategy to eradicate breast cancer. In addition, Loss of suppressors of Oct4 may be evaluated as a novel and powerful prognosis marker in the breast cancer.

    Lay Abstract:
    Genetic and Chemical Screens for Suppressors of Oct4 Expression in Breast Cancer Currently, the fundamental problem of breast cancer treatment is the recurrence of drug resistant cancer even after an impressive clinical remission. Recent studies identified that, indeed, only a small population of breast cancer cells are tumorgenic. These cells possess stem-like properties, which are usually resistant to traditional chemotherapeutic and radiation therapies and lead to the recurrence of cancer locally, regionally and distantly. This ?breast cancer stem cell? concept reveals the new paradigm of treatment. To eradicate breast cancer, it is necessary to effectively target these cells. Oct4 is a master regulator in maintaining stem cell properties in embryonic development. Re-expression of Oct4 has been found in the small population of breast cancer cells, which are able to cause tumor formation in somatic cells. However it is not found in normal tissues. Therefore, suppressing Oct4 expression will provide an effective strategy to target this small population of cells with minimized toxicity to the patients. However, what the intrinsic and extrinsic suppressors of Oct4 expression are remains an outstanding question. To answer this, we will employ two high-throughput genetic and chemical screens to systematically identify (1) what are intrinsic suppressors of Oct4 expression, and (2) what are potential drugs that can inhibit Oct4 expression and/or specifically kill Oct4 expressing cells. In the first specific aim, an enhanced retroviral mutagenesis (ERM) system will be applied to identify genes that will reduce Oct4 expression if over-expressed. In the second specific aim, an imaging-based assay system will be utilized to identify drugs that can specifically turn off Oct4 expression and/or kill Oct4 positive cells by screening chemical libraries containing a large collection of chemical compounds and FDA-approved drugs. The proposed research is based on the new paradigm of breast cancer treatment by targeting cancer cells with stem cell properties. Combining two advanced high-throughput screens to systematically identify intrinsic and extrinsic suppressors of Oct4 expression will result in a better understanding of how breast cancer cells acquire stem cell like properties. More importantly, results from proposed studies will have tremendous clinical implications in reducing mortality of breast cancer. Oct4 is not expressed in normal cells and its re-expression in breast cancer cells contributes to their stem cell like properties, which are the underlying mechanism for cancer recurrence. Therefore, targeting Oct4 will provide a general, effective and selective novel therapeutic strategy to achieve recurrence-free survival and long-term success in breast cancer treatment, regardless of subtypes of breast cancers. The chemical library containing FDA-approved drugs allows us to identify clinical drugs with novel effects on the suppression of Oct4. This will shorten the time frame for clinical application. Furthermore loss of Oct4 suppressors might also serve as a prognosis marker to evaluate the risks of cancer recurrence.