Defining YAP/TAZ-dependency in human breast cancer cells
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OVERVIEW: Hyperactivation and amplification of the oncogenic transcriptional co-factors YAP and TAZ are common in breast cancer. However, it is unknown if breast cancer cells are dependent on YAP/TAZ for growth and survival. In addition, key transcriptional targets of YAP/TAZ that enable breast cancer growth have yet to be defined. To address these unresolved questions, we will define YAP/TAZ-dependencies across a large cohort of breast cancer cells and generate gene expression signatures for both YAP/TAZ-dependent and YAP/TAZ-independent lines. We aim to identify YAP/TAZ-target genes that are essential for the growth and survival of YAP/TAZ-dependent breast cancer cells. This approach may reveal genetic dependencies in breast cancer that can then be therapeutically exploited. METHODS: A comprehensive cohort of breast cancer cells (45 cell lines) was obtained from the American Type Culture Collection (ATCC). The majority of time allotted for this thesis was spent culturing and expanding the cell lines. Five complete breast cancer cell line libraries were successfully generated and annotated. These libraries will be a useful resource for the Boston University School of Medicine Cancer Research Community. Protein and RNA extracts were collected from all cell lines. RNA extraction was performed in all cell lines with the Qiagen RNase Kit as per the manufacturer’s instructions. Protein extracts were collected from the cell lines with RIPA lysis buffer. Protein lysates were then run on an acrylamide gel and the relative abundance of YAP and TAZ was quantified. RNA extracts were sent for microarray analysis to obtain gene expression profiles. Cell lines were also fixed and stained for YAP and TAZ at subconfluence (50%) and confluence (90%) and visualized through immunofluorescence to assess the relative subcellular localization of YAP and TAZ. RESULTS: Our results indicate that YAP/TAZ levels and activity are highly variable across breast cancer cell lines. Seven cell lines were found to overexpress only YAP, nineteen cell lines were found to overexpress only TAZ, and two cell lines (BT-474 and HCC 1599) were found to overexpress both YAP and TAZ. Two cell lines (MDA-MB-134-VI and DU4475) had negligible protein expression levels of YAP/TAZ. We were also able to identify a subset of cells as being resistant to Hippo pathway activation, as seen in MCF 10A, MCF 10F, and MCF-12A cells, which maintained nuclear YAP and TAZ even under confluent conditions, and with MDA-MB-231 cells, which maintained only nuclear YAP under confluence. Given the importance of YAP and TAZ in cellular proliferation and survival, these results suggest that these Hippo pathway inactive cell lines may be dependent on YAP and TAZ for survival, which will be assessed at a future time point. We plan to complete our analysis of the subcellular localization of YAP and TAZ for all 45 breast cancer cell lines. Microarray profiling and gene expression signature analysis of all 45 cell lines are also ongoing. DISCUSSION: We surmise that increased levels/activity of YAP/TAZ will predict increased dependency on these oncogenes for growth and survival. This prediction will be directly tested by assessing cell viability following YAP/TAZ knockdown experiments. We also hypothesize that YAP/TAZ-dependent cells will be dependent on the transcription of specific YAP/TAZ target genes for survival. Current work detailed in this thesis will form the foundation for future work focusing on therapy-relevant YAP/TAZ target genes that are critical to breast cancer pathogenesis and disease progression. Our long-term aim is to identify pharmacologically-tractable YAP/TAZ target genes with the ultimate goal of finding novel chemotherapeutics that will improve prognosis for breast cancer patients.