Date of Degree
Access restricted until 08/31/2018
MS (Master of Science)
Sarcomas are cancers of mesenchymal origin. Though they comprise 15-20% of childhood cancers, and have a 5-year survival rate of 16% for metastatic disease, few targeted therapies exist, and the underlying mechanisms of their development are poorly understood. Transcriptional coactivators TAZ and YAP promote cell growth and proliferation, and are constitutively activated in a number of carcinomas. Accompanying TAZ/YAP activation in these cancers is decreased expression of Hippo pathway kinases MST1/2 and LATS1/2. As the Hippo pathway is the primary negative regulator of TAZ/YAP, this provides a potential mechanistic explanation for constitutive TAZ/YAP activation. TAZ and YAP are also thought to play a prominent role in sarcomagenesis, as TAZ-CAMTA1 and YAP-TFE3 gene fusions are the specific initiating events leading to formation of epithelioid hemangioendothelioma (EHE), a vascular sarcoma. However, the mechanisms causing constitutive activation of wild-type TAZ/YAP in sarcomas have not been well-characterized.
The purpose of this study was to determine if Hippo pathway dysregulation occurs in sarcomas with constitutively active, wild-type TAZ/YAP, as well as the mechanisms by which this regulation is lost. We also investigated whether TAZ/YAP could be therapeutically targeted in sarcomas using verteporfin, a small-molecule inhibitor of the TAZ/YAP-TEAD interaction. To address these questions, sarcoma cell lines and patient clinical samples were utilized. Using 159 patient tumor sections, we constructed a tissue microarray, stained for activated (nuclear localized) TAZ/YAP, and Hippo kinases MST1/2 and LATS1/2. A majority of sarcomas contained activation of both TAZ and YAP, while significant decreases in MST1/2 and LATS1/2 expression were observed. Results indicated a majority of tumors which stained positively for nuclear-localized TAZ/YAP also contained loss of expression of at least one of the four kinases evaluated.
All cell lines evaluated via immunofluorescence also had constitutively active (nuclear) TAZ/YAP when grown to confluence, which suggested they were no longer being negatively regulated by the Hippo pathway. In ~50% of lines, protein loss of MST1/2 and LATS2 occurred and mRNA expression of MST1 and MST2 was notably decreased in ~50%, although loss of LATS1 and LATS2 was minimal. Potential mechanisms which could account for Hippo kinase loss were next investigated. It was found that protein degradation diminished MST2 in 25% of cell lines. Regulation by epigenetic modifications was also investigated; hypermethylation accounted for slightly reduced MST1/2, while 67% of lines had histone deacetylation in both kinases. Whether TAZ/YAP can be therapeutically targeted using verteporfin was tested; treatment significantly inhibited anchorage-independent growth, proliferation, and TAZ/YAP transcriptional activity in sarcoma cell lines.
Our results collectively demonstrate TAZ/YAP activity can be targeted in sarcomas with verteporfin, and their constitutive activation is due to loss of MST1/2 and LATS2 kinase expression through protein degradation, histone deacetylation, and promoter hypermethylation. Such findings enhance our current comprehension of the molecular events which promote sarcomagenesis; this knowledge also opens up the possibility of creating targeted pharmacological interventions.
xi, 83 pages
Includes bibliographical references (pages 79-83).
Copyright © 2017 Sarah Lynne Hall