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CXCR:4 Wnt Axis in Chemoresistance of Breast Cancer Stem Cells
Tumor Cell Biology I
BACKGROUND: Cancers originating from stem cells are suggested to be highly metastatic and chemoresistant compared to cancers originating from committed progenitor or mature cells. However, markers that can distinguish cancers based on nature of cancer-initiating cell are yet to be characterized. We have identified a chemoresistant subpopulation of breast cancer stem cells based on the expression of CXCR4 and CD44 and CD24. CXCR4+/CD44+/CD24-(CXCR4+ progenitor) cells were resistant to the chemotherapy drug docetaxel compared to CXCR4-/CD44+/CD24-(CXCR4- progenitor) cells. Microarray analysis revealed specific overexpression of TCF4 and Nr-CAM (Wnt pathway molecules) and Contactin 1 (Notch pathway) in CXCR4+ progenitor cells. Because CXCR4 is expressed in mammary stem cells and Wnt/Notch pathways mediate stem cell self-renewal and homing in bone marrow microdomains, selective overexpression of components of these pathways in CXCR4+ progenitor cells reflects stem cell origin of these cancer cells. HYPOTHESIS: Wnt (TCF4):CXCR4 axis mediates self-renewal and stem-specific gene expression in cancer stem cells, which contributes to metastasis and drug resistance. SPECIFIC AIMS: 1) Establish a molecular link between the CXCR4 and TCF4 and determine the role of TCF4 in self-renewal and chemoresistance in vitro. 2) Investigate the role of TCF4 in metastasis and chemoresistance in vivo. STUDY DESIGN: Aim 1: Asymmetric division of CXCR4+ progenitor cells will be evaluated by BrdUrd-Hoechst quench assay. CXCR4+ progenitor cells expressing a dominant negative mutant of TCF4 or TCF4 shRNA, and CXCR4- progenitor cells overexpressing TCF4 will be tested for asymmetric division, sensitivity to docetaxel in vitro and to identify TCF4-dependent stem cell-enriched genes. Aim 2: Mammary fat pad-adjuvant model and intracardiac model in nude mice will be used for in vivo studies with cells derived in aim 1. The first model will evaluate the role of TCF4 in lung metastasis from the mammary fat pad and the response of lung metastasis to chemotherapy. The second model will evaluate the role of TCF4 in multiorgan metastases and response of these metastases to chemotherapy. Potential outcomes: This study will validate CXCR4:TCF4 as markers that identify stem cell nature of drug resistant cancer cell subpopulation. If TCF4 proves to be a critical player in drug resistance, inhibitors of TCF4, such as the nitric oxide releasing aspirin, can be tested as chemosensitizers in future.
BACKGROUND: Breast cancer deaths are mostly due to the failure of current therapy to eliminate metastatic disease. Recently it is suggested that cancers originating from adult tissue stem cells are highly metastatic and are resistant to therapy. It is proposed that cancer stem cells undergo asymmetric division i.e., give rise to one drug-sensitive daughter cell and other chemotherapy-resistant cell that behaves similar to parental stem cell. The challenge is to isolate such cancer stem cells and to identify unique proteins that are involved in metastasis, asymmetric division and drug resistance. We have identified a specific subpopulation of breast cancer cells that are resistant to the chemotherapeutic drug docetaxel. These cells express the cell surface molecule CXCR4 and correspond to breast cancer progenitor/stem cells based on the expression pattern of CD44 and CD24 (CD44+/CD24-). Interestingly, we observed specific overexpression of genes in two signaling pathways known to be involved in asymmetric division of adult stem cell, Wnt (TCF4) and Notch (Contactin 1), in CXCR4+ stem cells. HYPOTHESES: CXCR4+ breast cancer stem cells utilize Wnt pathway for asymmetric division, metastasis and chemotherapy resistance. Inhibiting the activity of Wnt pathway molecule TCF4 should reduce asymmetric division, reduce metastasis and reverse resistance to chemotherapy. SPECIFIC AIMS: 1) Establish a molecular link between CXCR4 and the Wnt pathways and determine the role of this axis in asymmetric division and chemoresistance in vitro. 2) Investigate the role of TCF4 in metastasis and chemoresistance in vivo. STUDY DESIGN: For aim 1, we will inactivate TCF4-mediated signaling in CXCR4+ cancer stem cells by dominant negative mutants of TCF4 or siRNA against TCF4 and analyze them for asymmetric division and sensitivity to chemotherapy. For aim 2, we will use a nude mouse model to study the role of TCF4 in metastasis to lungs and bones and determine whether metastases with reduced TCF4 activity are more susceptible to chemotherapy. POTENTIAL OUTCOMES: This study will prove whether CXCR4+ progenitor cells correspond to the most primitive cancer stem cells that are resistant to treatment and whether that resistance is due to Wnt/TCF pathway. A number of avenues are being pursued to disrupt TCF4 signaling. One of the promising drugs is the nitric oxide releasing-aspirin, which will be evaluated in future for overcoming chemotherapeutic resistance of breast cancer stem cells.