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Dissertation
Elucidating the mechanisms of EHE transformation
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2024
DOI: 10.25820/etd.007683
Abstract
The Hippo signaling pathway regulates many key cellular processes including cell growth, apoptosis, and differentiation. The downstream effectors of the Hippo pathway, TAZ (transcriptional coactivator with PDZ-binding motif) and YAP (yes associated protein), are transcriptional coactivators that need to complex with Tea Domain (TEAD) family of transcription factors to drive their transcriptional program. This pathway is disrupted in many cancers, including sarcomas, leading to hyperactive TAZ and YAP. One mechanism TAZ and YAP can avoid regulation is by chromosomal translocation events resulting in the expression of novel chimeric proteins. Specifically, a TAZ-CAMTA1 or YAP-TFE3 fusion protein drives the vascular sarcoma epithelioid hemangioendothelioma (EHE). Understanding the mechanisms by which these fusion proteins drive cellular transformation may lead to better therapies for EHE, and potentially extend to other cancers that have activated TAZ or YAP.
In chapter 2, I focus on the generation and characterization of the first human EHE extended primary cell cultures. I demonstrated expression of the TAZ-CAMTA1 fusion protein by qRTPCR, expression of CAMTA1 by single-cell RNA seq, and expression at the protein level by probing for TAZ and observing a shift in molecular weight to what is expected for the fusion protein. I found that the EHE primary cell cultures proliferate faster than primary endothelial cells in 2D culture and drive anchorage-independent growth in soft agar and poly-HEMA. Analysis of genomic alterations in the cell cultures showed a low mutational burden, corroborating previously established literature. Upregulated pathways defined by RNA-seq in EHE tumors are conserved in primary cell cultures by RNA-seq. Additionally, I have shown that the extended primary cell cultures have not spontaneously immortalized and senescence around passage 20, suggesting other alterations besides TAZ and YAP fusion protein expression are needed to drive full transformation.
In chapter 3, I sought to identify other cofactors necessary for EHE cellular transformation. I found that topoisomerase II (TOP2) is required for YAP-TFE3 and TAZ-CAMTA1 mediated anchorage-independent growth. This stems from a high-throughput screen of 1,000 FDA approved compounds in which 4/6 that reduced spheroid size by the greatest amount are TOP2 inhibitors. Follow-up studies by genetic knockdown of TOP2 show a decrease in anchorage-independent growth, a key phenotype driven by the fusions, but no decreases in 2D proliferation. TOP2 has a variety of cellular functions including alleviating supercoiling that occurs during DNA transcription and replication. Emerging literature suggests that TOP2 aids in regulating chromatin topological associated domains, in part through chromatin looping, and impacts transcription. While TOP2 inhibitors are used clinically to treat a variety of cancers including sarcomas, they are used judiciously because of cardiotoxic side effects.
A greater understanding of the mechanisms driving EHE transformation is required to generate better therapies. The work presented in this thesis describes and characterizes the first human EHE extended primary cell cultures. I also identified topoisomerase II as a cofactor and potential therapeutic vulnerability in EHE, likely by regulation of YAP-TFE3 and TAZ-CAMTA1 transcription. Overall, the findings discussed in this thesis provide rationale for exploring new therapeutic options to target EHE and describe a new cell-based model to facilitate new discoveries.
Details
- Title: Subtitle
- Elucidating the mechanisms of EHE transformation
- Creators
- Nicholas Philip Scalora
- Contributors
- Munir R. Tanas (Advisor)Benjamin W. Darbro (Committee Member)Rebecca D. Dodd (Committee Member)David J. Gordon (Committee Member)Dawn E. Quelle (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biomedical Science (Molecular Medicine)
- Date degree season
- Summer 2024
- Publisher
- University of Iowa
- DOI
- 10.25820/etd.007683
- Number of pages
- xii, 93 pages
- Copyright
- Copyright 2024 Nicholas Philip Scalora
- Language
- English
- Date submitted
- 07/22/2024
- Description illustrations
- Illustrations, tables, graphs, charts
- Description bibliographic
- Includes bibliographical references (pages 87-93).
- Public Abstract (ETD)
Epithelioid hemangioendothelioma (EHE) is a sarcoma that arises from blood vessels and can occur anywhere throughout the body. EHE is caused by mutually exclusive fusion proteins involving end effectors of the Hippo signaling pathway, TAZ and YAP. A TAZ-CAMTA1 fusion protein accounts for ~85% of EHE cases, while a YAP-TFE3 fusion protein accounts for the remaining 15%. In normal conditions, TAZ and YAP are negatively regulated by Hippo to prevent aberrant transcription that can lead to cancer. The fusion events result in the loss of the key regulatory residues within TAZ and YAP and are therefore largely unencumbered to drive their oncogenic transcriptional program. Currently, there is no consensus on the best therapy for EHE patients, partially due to minimal model systems to study this cancer.
In this thesis, I have developed and characterized the first human EHE extended primary cell cultures that can serve as a platform for further mechanistic studies in attempts to find more effective therapies. I have also shown that YAP-TFE3 and TAZ-CAMTA1 are particularly susceptible to a class of chemotherapy drugs called topoisomerase II inhibitors. Topoisomerase II is a critical enzyme that functions to remove DNA tangles and supercoils that accumulate in the cell. This susceptibility highlights potential combination therapies that could be more effective in treating EHE. The fusion proteins can serve as a model to study hyperactive TAZ and YAP which occurs in over half of sarcomas, so the findings of this thesis may be applicable in other cancers.
- Academic Unit
- Biomedical Science Program
- Record Identifier
- 9984698151802771
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