The goal of this proposal is to determine if knocking down Maternal Embryonic Leucine zipper Kinase (MELK) expression will inhibit mammary tumor formation in vivo. MELK is overexpressed in human breast cancers compared to normal tissue. Knocking down levels of MELK transcript with siRNA inhibits the in vitro growth of several types of tumors, including mammary tumors. However, the role of MELK in mammary tumorigenesis in vivo has not been demonstrated. We will use a competitive transplantation assay to test our hypothesis that MELK function is required for mammary tumorigenesis in vivo.
Aim1. Determine if MELK function is required for development of Polyoma Middle T (PyMT) induced mammary tumors in vivo. We will isolate mammary tumor cells from MMTV-PyMT mice and knock down MELK transcripts using lentivirus expressing MELK shRNA and the red fluorescent protein mCherry. Control cells will be infected with scrambled shRNA co-expressed with eGFP MELK shRNA-infected tumor cells will be admixed with control cells and transplanted into syngenic cleared mammary fat pads to generate tumors. The resulting tumors will be analyzed for the presence of donor cells infected with MELK shRNA. We expect to find no/few such cells if MELK shRNA inhibits tumor development in vivo.
Aim 2 : Determine if MELK function is required for development of Wnt-induced mammary tumors in vivo. Mammary tumors induced in the MMTV-Wnt model are significantly more heterogeneous than are PyMT tumors and are more typical of the majority of human breast cancers. We will use MMTV-Wnt1 transgenic mice to test the MELK hypothesis in this model exactly as described for the PyMT model. In both cases we will: 1) establish limited dilution transplantation efficiency of primary tumors;2) isolate tumor cells, infect them with MELK shRNA or scrambled control shRNA lentivirus and transplant them into cleared mammary fat pads;3) analyze tumors formed in transplanted animals using a combination of fluorescence microscopy, FACS and Q- PCR for the presence of MELK shRNA (mCherry/red) infected cells and endogenous MELK transcripts. There is a crucial need for potent inhibitors of breast cancers. If MELK function is a prerequisite for mammary tumor growth in vivo, mouse tumor cells and animal models can be used to screen and validate compounds that specifically inhibit MELK function in mammary tumor models (e.g., specific kinase inhibitors that have been developed in the PI's laboratory). Given the very high homology between human and mouse MELK kinase domains, compounds validated in a mouse model will be the leads for developing clinically relevant inhibitors to target human breast cancer. PROJECT NARRATIVE: The lifetime risk of breast cancer for women in the USA is 1 in 8, with about 200,000 new cases diagnosed each year. There is a crucial need for potent inhibitors of breast cancers. A candidate gene, Maternal Embryonic Leucine zipper Kinase (MELK), is overexpressed in most breast cancers, particularly in aggressive, poorly differentiated tumors. Despite indications that knocking down MELK inhibits tumor cell growth in culture, evidence for an in vivo role for MELK in tumorigenesis is missing. Our hypothesis is that MELK function is required for mammary tumorigenesis in vivo.
In Aim 1 we will use one of the best established mammary tumor models, Polyoma Middle T (PyMT) induced tumors.
In Aim 2 we will take advantage of the more recently established Wnt-induced tumor model. We have shown that MELK expression marks PyMT tumors in vivo and that knocking down MELK expression inhibits growth of a mammary tumor cell line in vitro. PyMT-induced tumors are homogeneous and can be very efficiently transplanted in vivo making the PyMT model the most favorable one in which to test our hypothesis. PyMT-induced tumors resemble a subset of human breast tumors. However, other human tumors are more heterogeneous and better captured by the Wnt-induced tumor model. Wnt-induced tumors are heterogeneous and likely initiated in the progenitor compartment, thus retaining some features of mammary gland progenitor cells. Our preliminary data suggest that mammary gland end buds containing those progenitor cells express high levels of MELK. Thus, MELK expression may be retained in transformed cells and play a functional role in tumor initiation in Wnt1-induced mammary tumors. In both mouse tumor models we will perform a similar set of experiments. First, we will establish the transplantation efficiency of primary tumors;second, we will infect tumors with inhibitory small hairpin RNAs (MELK shRNAs) or a scrambled control shRNA and transplant those tumors into mammary fat pads. Third, we will analyze tumors formed in transplanted animals using a combination of fluorescence microscopy and flow cytometry to determine if donor cells infected with MELK shRNA form tumors. If MELK shRNA inhibits tumor formation in vivo we expect not to find these cells. If MELK function is a prerequisite for mammary tumor growth in vivo, mouse tumor cells and animal models can be used to screen and validate compounds specifically inhibiting MELK function in mammary tumor models (e.g., specific kinase inhibitors that have been developed in the PI's laboratory). Given the very high homology between human and mouse MELK kinase domains, compounds validated in a mouse model will serve as the leads for developing clinically relevant inhibitors to target human breast cancers.
| Hebbard, Lionel W; Maurer, Jochen; Miller, Amber et al. (2010) Maternal embryonic leucine zipper kinase is upregulated and required in mammary tumor-initiating cells in vivo. Cancer Res 70:8863-73 |
