DEPARTMENT OF DEFENSE - CONGRESSIONALLY DIRECTED MEDICAL RESEARCH PROGRAMS

Breast Cancer Stem Cells: Multidrug Resistance and Survival in the Bone Marrow Niche

Principal Investigator: HAUSCHKA, PETER V
Institution Receiving Award: CHILDREN'S HOSPITAL, BOSTON
Program: BCRP
Proposal Number: BC050864
Award Number: W81XWH-06-1-0422
Funding Mechanism: Idea Award
Partnering Awards:
Award Amount: $477,031.60


PUBLIC ABSTRACT

Stem cells are unique in their property of endless self-renewal. This is essential for the healthy development of normal embryos and for the repair and renewal of adult tissues. But in cancer, stem cells are now theorized to be the central problem that prevents permanent eradication of the disease in a patient. In breast cancer, the cancer stem cell arises from mutations in a normal regenerative breast epithelial stem cell. As this cell divides rapidly and produces a tumor mass, its progeny become heterogeneous by a process of asymmetric cell division. Some of the progeny, and all of the stem cells, produce proteins called ¿efflux pumps¿ that are critical for progression of breast cancer. Efflux pumps sit in cell membranes and use chemical energy from ATP to grab molecules or ions from inside the cell and push them to the outside of the cell. The most important of these pumps, ABCG2, is also called "breast cancer resistance protein" (BCRP) because it was first discovered to be abundant in drug-resistant forms of breast cancer. ABCG2 pumps out organic chemotherapeutic drugs such as topotecan, etoposide, doxorubicin--favored second-line options for breast cancer. Our hypothesis is that breast cancer is driven by stem cells that have intrinsically high levels of the ABCG2 pump. This self-renewing, proliferative breast cancer stem cell population accounts for malignant cancer progression and chemotherapeutic resistance. Thus a major therapeutic dilemma exists, since almost any combination of drugs would never be able to kill these cancer stem cells.

This Project takes an innovative approach by studying a newly discovered feature of breast cancer, the cancer stem cell, from a novel perspective that links these cells to bone marrow, the favored site for metastasis. We recently discovered that these stem cells have highly elevated expression of five genes that could be critical for breast cancer progression: ABCG2 and MRP3 (drug efflux pumps), RANK (a receptor that blocks cell death by interacting with RANKL, a protein heavily produced by osteoblasts), and Oct4 and Nanog (transcription factors that control the genes required for self-renewal of stem cells). Efflux pump expression in breast cancer stem cells makes these cells able to resist death in the face of almost every therapeutic agent. Moreover, these are the cells that cause relapse by reforming new tumors after each round of surgery, radiation, and failed chemotherapy. We now have a procedure ("flow cytometry") to specifically purify the breast cancer stem cells based on sensitive, specific measurement of the amount of efflux pump activity and other marker proteins in each single cell of a cell line or tumor. This allows the stem cells of breast cancer to be studied for their unique properties and pathways for the first time. Early data confirm the validity of our approach. 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 planned study has three objectives. First we will isolate and characterize the rare stem cells from model human breast cancer cell lines. To do this, ABCG2 and RANK are tagged with two different colored fluorescent antibodies, allowing cells that are bright for both colors to be sorted and collected by flow cytometry. These are the rare putative cancer stem cells that we compare with their abundant, but dim, non-stem cell siblings for properties of efflux pump activity, gene expression profile, and self-renewal. Second, the increased malignant behavior of the breast cancer stem cells will be studied by assay of proliferation, cell death caused by cytotoxic drugs, drug resistance, self-renewal, and supportive interaction with the osteoblast "niche." Third, we will inject breast cancer stem cells or their non-stem-cell counterparts into the bone marrow of experimental mice to determine how their expression of critical genes affects tumor progression over a 12-week interval. We employ well-characterized human breast cancer cell lines with graded malignant properties to study these questions in cell culture and in the mouse model. Our hope is to translate this approach to breast cancer stem cells isolated from primary human cancers. Molecular tools will be used to selectively silence ABCG2, RANK, Oct4, and Nanog in different pools of stem cells in order to prove the requirements for each gene in drug-resistant cell survival, rapid cell division, perpetual renewal of the stem cell, and efficient tumor formation in the living animal.

We are confident that the outcome of our experiments will serve as a foundation for more effective therapy of breast cancer. While scientists are aware that many cancer cell subtypes with subtle differences exist in every breast tumor mass of a patient, treatments currently assume that all the cells have unlimited proliferative potential and ability to metastasize. If breast cancer stem cells can be identified, then the targeted elimination of only these cells might be sufficient to destroy the cancer and obtain long-term remission. One of our planned experiments has major therapeutic implications for blocking the self-renewal of breast cancer stem cells by inhibiting their high expression of Oct4 and Nanog. Other studies will establish strategies for combined therapies with cytotoxic drugs, efflux pump inhibitors, and inhibitors of osteoblast/bone marrow support, with the goal of specifically targeting and killing even the most refractory breast cancer stem cells in patients.