Maternal Embryonic Leucine Zipper Kinase in Mammary Tumors

Institution: The Burnham Institute for Medical Research
Investigator(s): Robert Oshima, Ph.D. -
Award Cycle: 2008 (Cycle 14) Grant #: 14IB-0082 Award: $286,500
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2008)

The maternal leucine zipper kinase (MELK) gene is a potential marker of proliferating mammary epithelial progenitor cells that are highly expressed in multiple human cancers, including human breast cancer. Using a genetically engineered mouse model, we have observed increased expression of Melk in restricted cells of the normal mammary gland and in tumor cells arising due to the expression of activated Neu/ErbB2, Polyoma middle T antigen and Wnt 1 oncogenes. A new targeted mutation of the Melk gene is now available to determine if Melk expression is functionally important to mammary tumor cells caused by an oncogene that uses the same signaling pathways as ErbB2/ErbB3 oncogenes.

Our objective is to inactivate Melk only in mammary tumor cells to evaluate its importance strictly in cancer cells. We will combine three different genes that have each been engineered for specific reasons. First, the Melk gene has been changed so that it can be permanently inactivated by the action of a protein called Cre recombinase. The second gene codes for the Cre protein and has been engineered to be expressed only in mammary cells and only at puberty. The third gene is a silent gene that expresses a powerful cancer causing protein but only when Cre recombinase removes an inhibitory sequence. Thus in the presence of Cre, this gene will be activated to express the oncogene that causes tumors to form. In the same cell, Cre will inactivate the Melk gene. By comparing mice that either have or do not have the Melk protein, we will be able to determine if Melk is critical for the start and continuation of tumors in the mammary gland.

This project will permit us to evaluate the effect of Melk in only tumor cells, independently from the neighboring normal cells (stroma) that do not develop into tumors, just as the processes occur in patients. This strategy takes advantage of one the common problems of expressing Cre recombinase in animals. That problem is that rarely is Cre expressed in every cell of a particular tissue. This problem of incomplete expression in all cells of a tissue complicates and in some cases compromises the expected results. In our case this will actually be an advantage, permitting us to evaluate the importance of Melk only in prospective tumor cells.

Final Report (2010)

This project was to determine the functional importance of the maternal embryonic leucine zipper kinase (Melk) on breast cancer. We used a mouse in which the Melk gene has been altered by removing a portion of the gene that codes for key regions of the protein. This altered gene is capable of generating an altered Melk protein, but this protein is unable to perform the chemical reaction that the native protein does. This kinase activity involves using ATP to place a phosphate group on a protein substrate. The mutant protein is unable to perform this function.

In our first studies, we combined the genes of this mouse that carries this Melk gene deletion with another mouse that develops mammary tumors due to the expression of a powerful oncogene in mammaryepithelial tissues. We compared female mice that have two copies of the defective Melk gene with mice with normal Melk genes or only one defective gene. All of the animals also carry the oncogene that causes mammary tumors in about 3 months. We found that the defective Melk gene has no impact on tumor number, size, appearance,weight, or frequency of metastasis. Thus the Melk kinase activity is not required for mammary tumors in this model.

However, additional experiments using shRNA (short hairpin RNA) knockdown of the Melk decreased the ability of cultured mammary tumors to both form tumors in vivo and form tumorspheroid colonies in cell culture. Melk shRNA decreased tumor frequency by six fold. This study suggests that Melk protein, but not kinase activity, may be important for mammary tumor formation.