High-grade serous carcinoma (HGSC) is the most common subtype of ovarian cancer. Many of these tumors are now believed to originate in the tips of the fallopian tubes. The cells in the fimbria that are destined to become HGSC can be readily identified under the microscope by using an antibody that stains a protein called PAX8. Both normal fallopian tube cells and HGSCs share this protein. Previous work showed that PAX8 is essential for the development of normal fallopian tubes in mice. PAX8 is also needed to maintain growth of HGSCs.
Because of the central role for PAX8 in fallopian tube and HGSC biology, we hypothesize that PAX8 would be a good target for new cancer therapies. To achieve this goal, we need to understand how PAX8 functions as a transcription factor. Our proposed study will attempt to identify the protein partners of PAX8 that contribute to its activity. By characterizing these interactions, we hope to identify those that are critical to the function of PAX8 in ovarian cancer cells.
We will use two approaches to identify PAX8 partners. The first approach relies on an antibody that recognizes PAX8. We will use this antibody to affinity-purify PAX8 from benign fallopian tube cells and from ovarian cancer cell lines. We will then apply mass spectrometry to identify all the proteins that are co-purified with PAX8. One of our goals here is to determine whether PAX8 has different partners in normal cells versus cancer cells. In our second approach, we will engineer a form of PAX8 that can be easily purified from cells. This form of PAX8 will have two small ‘tags’ added to either end of the protein. We can use antibodies against these tags to readily purify the PAX8 complex. Again, interacting proteins will be identified by mass spectrometry. Once we have validated the interacting proteins as bona fide PAX8 partners we will determine whether they functionally contribute to the transcription activity of PAX8. We will achieve this by using a platform that depends on the DNA binding activity of PAX8 to turn on the expression of a gene called luciferase. The levels of luciferase in a cell can be readily monitored. Cells that express PAX8 will turn on the luciferase gene. Cells that do not express PAX8 will not express luciferase. With this PAX8-luciferase system, we will be able to ask whether the PAX8 protein partners that we identified contribute to the ability of PAX8 to turn on the expression of luciferase. The expression of each protein partner will be knocked down or eliminated in the cells and the expression of luciferase will be determined. If a PAX8 protein partner is essential for PAX8 activity, we will lose luciferase expression when that protein is knocked down. Such a result would support an important role for that protein in PAX8 function. Our studies will enable us to begin selecting which proteins are essential for PAX8 transcription activity. These proteins will be important targets for new cancer therapies.