Research Grants Awarded
Skeletal Metastasis and the Unique Role of Breast Cancer Stem Cells
Tumor Cell Biology I
Cancer stem cells represent a new paradigm for progression of breast cancer to its bone-metastatic, drug-resistant, untreatable endpoint. Asymmetric division of the stem cell cohort may explain the phenotypic heterogeneity characteristic of recurrent breast cancer. ABC-type m embrane efflux pumps are centrally involved in drug resistance. ABCG2/BCRP effluxes etoposide, doxorubicin, and other drugs, and is a defining marker of stem cells. In the bone-metastatic human breast cancer line MDA-MET, cell sorting has revealed a “side population” of ABCG2+ stem cells with highly elevated expression of transcription factors linked to self-renewal (Oct4, Nanog), and a second pump (ABCB4/MDR3). HYPOTHESIS: Bone metastasis is seeded by stem cells with uniquely high expression of genes for self-renewal (Oct4, Nanog), drug efflux (ABCG2, MDR3), and bone homing/survival (RANK). The ABCG2+/RANK+ subset of breast cancer stem cells metastasize efficiently in bone marrow because of survival signaling from RANK binding its ligand RANKL on resident osteoblast membranes. OBJECTIVE: Purify and characterize the RANK+ breast cancer stem cells from cell lines and clinical metastatic pleural effusions to identify factors controlling malignancy, tumorigenicity, and metastasis. AIM 1) Characterize the phenotype and gene expression profile of purified stem cell populations, and test regulatory factors that promote self-renewal vs. differentiation. AIM 2) Establish the role of the osteoblastic niche in multidrug resistance and self-renewal by co-culture models of breast cancer stem cells and osteoblasts. AIM3) Determine the tumorigenicity of breast cancer stem cells (vs. non-stem cells and siRNA-silenced cells) by injection into the bone marrow compartment of mice. DESIGN: Stable silencing of ABCG2, RANK, Oct4, or Nanog in stem cells will test regulatory control of gene expression, efflux pump activity, drug-resistance, self-renewal, and metastasis. OUTCOMES/BENEFITS: Our novel approaches for p rospective identification and analysis of the highly tumorigenic RANK+ stem cell phenotype in breast carcinoma will reveal genes/pathways regulating drug-resistant survival, and supportive interactions with osteoblasts relevant to metastasis. Because they drive tumor progression, direct therapeutic attack of the critical breast cancer stem cells through the targeting of efflux pumps, RANK/RANKL, and self-renewal machinery should be promising for breast cancer treatment.
Breast cancer stem cells continuously generate heterogeneous malignant cells and are a central obstacle to permanent eradication of disease in survivors. Identification of breast cancer stem cells should enable their targeted elimination, leading to long-term remission. Our novel perspective is to study drug-resistant survival of these stem cells in bone marrow, the favored metastatic site of breast cancer. The focus is a unique class of cancer stem cells that express RANK (a cell survival receptor activated in bone), and ABCG2/BCRP (breast cancer resistance protein; a stem cell marker and efflux pump for chemotherapeutic drugs ). These cells have a drug-resistant survival advantage in the initiation of bone metastasis. OBJECTIVE: Obtain detailed molecular profiling and characterization of RANK+ breast cancer stem cells as a strategic foundation for therapeutic targeting of bone metastasis. HYPOTHESIS: Bone metastasis is seeded by stem cells with uniquely high expression of genes for self-renewal (Oct4, Nanog), drug efflux (BCRP, MDR3), and bone homing (RANK). AIM 1) Isolate and characterize stem cells from human breast cancer cell lines and clinical metastatic pleural effusion samples. BCRP, RANK, and other markers tagged with fluorescent antibodies are sorted by multicolor flow cytometry. Cancer stem cell gene expression profiles and efflux pumps are compared with non-stem cells. AIM 2) Analyze malignant behavior of stem cells by assay of proliferation and self-renewal, death from cytotoxic drugs, and drug resistance . Assess cancer stem cell survival signals resulting from RANK binding to its partner RANKL on bone marrow osteoblasts. AIM 3) Inject breast cancer stem cells into bone marrow of mice to test the role of critical genes in tumor progression. DESIGN: Selective silencing of BCRP, RANK, Oct4, or Nanog in stem cells will prove the requirements for each gene in drug-resistant cell survival, perpetual self-renewal, and efficient metastasis formation. OUTCOMES/BENEFITS: We reason that detailed molecular and genetic knowledge of the breast cancer stem cells will provide a strategic base for developing new therapies that target and kill off this insidious stem cell reservoir. Our studies will establish rationales for combined therapies with cytotoxic drugs, efflux pump inhibitors, and inhibitors of osteoblast/bone marrow support, thereby targeting and killing even the most refractory breast cancer stem cells in patients.