A Candidate Marker of Mammary Tumor Initiating Cells

Institution: The Burnham Institute for Medical Research
Investigator(s): Alexey Terskikh, Ph.D. -
Award Cycle: 2006 (Cycle 12) Grant #: 12IB-0079 Award: $284,262
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2006)
Breast tumors are heterogeneous and may include hyperplasia, invasive carcinoma, reactive stroma, supportive blood vessels, and inflammatory cells. One key objective in breast cancer research is to identify appropriate cells to target for therapeutic intervention. Recently, a small proportion of breast tumor mass has been found to be able to produce tumors upon transplantation in animal models. These tumor initiating cells, also termed cancer stem cells, are likely to be the appropriate target cells. However, little is known about the molecular markers and functional targets operating in such cells. On the other hand, several mouse models of mammary tumors have been established to investigate the molecular and cellular mechanisms of breast cancers in vivo. However, no functional markers of tumor initiating cells have been described yet. Identification of such markers will allow us to focus the efforts on the relevant population of tumor cells, both in a research and clinical setting.

We propose that a new gene, called MELK (maternal embryonic leucine-zipper kinase) could be a functional marker for the tumor-initiating cells in breast cancer. MELK is expressed in a variety of tumor lines, including neuroblastomas, melanomas, hepatocellular and adenocarcinomas, papillomas, lymphoma, and germ cell tumors. The exact role of MELK in development and adults is uncertain, although similar kinases are associated with cell survival, cell cycle regulation, and RNA splicing functions. Our hypothesis is that MELK expression in mammary tumors labels the “cancer stem cells.”

We will use a transgenic mouse model to identify tumor-initiating cells in mammary tumors. We have generated mice where the Green Fluorescent Protein (GFP) is expressed under the control of a MELK promoter (gene regulatory) element. These transgenic mice will allow us to visualize and study the cells that express MELK/GFP markers. We will cross these “reporter mice” with another transgenic strain overexpressing oncogenes (e.g., PyMT or Wnt1) specific to mammary tissue. These double-transgenic mice allow us to investigate the MELK expression in mammary tumors and stem cells.

Final Report (2008)
Our goal is to determine if MELK (A full description of MELK with a discussion of its potential role in various cell biological contexts is found on-line: http://www.signaling-gateway.org/molecule/query?afcsid=A003002) is a functional marker of transplantable mammary tumor cells using Polyoma virus middle T antigen (PyMT) and Wnt1 mouse models of mammary tumors. For a discussion of Wnt1 see:
http://en.wikipedia.org/wiki/WNT1. Our hypothesis is that MELK expression defines the tumor initiating cells in mammary tumors. Our first objective is to determine if MELK/GFP-positive cells from MMTV-PyMT mammary tumors are transplantable tumor initiating cells in vivo. Our second objective is to determine if MELK/GFP positive cells from MMTV-Wnt1 mammary tumors are transplantable tumor initiating cells in vivo.

We have made significant progress in both directions. In particular, we have completed the animal breeding to obtain the MELK GFP xMMV-PyMT and MELk-GFPxMMTV-Wntl mice on pure FVB/N background. This is a critical step for the success of the transplantations into the recipient FVB/N mice (histocompatibilty of the donor and the recipient mice is a critical parameter for the immune rejections of normal cells and tumors). On the other hand we have performed additional flow cytometry (FACS) analysis to position the MELK-GFP expression within the stem cell and/or progenitor compartment (see results below). In parallel, in collaboration with the group of Robert Oshima at Burnham, we performed pilot transplantation of the normal mammary gland cells from MELK-GFP mice. We found that GFP+ (green) cells are more potent in reconstituting the mammary trees as compared to GFP- cells. These results corroborate our hypothesis that MELK/GFP labels normal mammary stem cells in addition to neoplastic (tumor) cells. We have focused on the MMTV-Wnt1 tumors because these tumors are more heterogeneous consisting of both myo and luminal compartments (only luminal in case of MMTV-PyMT tumors). The Wnt model is a more realistic representation of human breast cancers including the suggested presence of mammary tumor stem cells.

We have established the MMTV-Wnt1 X MELK-GFP mouse models and transplanted the High vs. Low cells into syngenic (genetically identical) recipients. We have performed limited dilutions experiment to determine the minimum numbers of MELK-GFP+ cells capable of transplanting the Wnt-1 tumors. We have established that as few as 10 MELK/GFP high cells in the Wntl primary tumors can efficiently give rise to the secondary tumors upon transplantation 5 out of 16 transplants) while MELK/GFP low cells have much lower tumor initiation rate (1/16). These experiments identify MELK as a prospective marker for mammary tumor stem cells. Because MELK catalytic function has been recently implicated in mammary tumorigenesis these results suggest that small molecule inhibitors of MELK (currently being developed in our laboratory) could be developed into clinically relevant drugs to inhibit the breast cancer progression in patients.