Dissertation
TAZ and YAP dysregulation in sarcomas
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2022
DOI: 10.25820/etd.006506
Abstract
Sarcomas are comprised of a diverse group of cancers with over 50 histological types, which are difficult to treat. In addition to lack of medical therapies, the mechanisms that drive sarcomagenesis remain to be fully understood. Current treatment modalities for localized disease, which has a survival rate of approximately 60%, often involves surgical resection. Depending on the size and location of the tumor, radiation may be employed prior to surgical removal. The 5-year survival rate for patients that develop metastatic disease is around 15-20%. Treatment for metastatic sarcoma is limited and often involves toxic chemotherapeutic agents, such as doxorubicin, a topoisomerase inhibitor, which can lead to severe cardiotoxic effects. Therefore, there is a critical need to identify actionable targets for therapeutic benefit in the treatment of sarcomas.We currently know that the Hippo tumor suppressor pathway plays a critical role in a majority of sarcomas. Downstream effectors of the Hippo pathway are two transcriptional co-activators, TAZ (transcriptional coactivator with PDZ-binding motif) and its paralogue YAP (yes associated protein). In sarcomas as well as other cancer types including breast, colorectal, ovarian, liver, pancreas, and thyroid cancer, this pathway is disrupted leading to a hyperactive TAZ and YAP oncogenic transcriptional activity. TAZ and YAP are activated in >50% of sarcomas and many of these sarcomas exhibit loss of expression of the Hippo tumor suppressor kinases, MST1/2 or LATS 1/2. Importantly, activating point mutations in TAZ and YAP are rare and alterations of these proteins are often involved in gene rearrangements resulting in oncogenic fusion proteins.
The vascular sarcoma, epithelioid hemangioendothelioma (EHE), is defined by the gene fusions TAZ-CAMTA1 (TC) and YAP-TFE3 (YT) which are observed in approximately 85% and 15% cases, respectively. The fusion proteins contain the N-terminus of TAZ and YAP, including their TEAD binding domains, fused in frame to the C-terminus of CAMTA1 and TFE3, respectively. CAMTA1 and TFE3 contain transactivating domains as well as other protein-protein interaction domains leading us to hypothesize that the C-terminus of TAZ-CAMTA1 and YAP-TFE3 interact with novel proteins that augment the oncogenic activity of what is typically observed for wild-type TAZ and YAP.
Discussed in Chapter 2, I explore the oncogenic properties of TC and YT in vitro and in vivo. I demonstrate that YT is capable of transforming cells in vitro to promote anchorage independent growth, and this is dependent on components of both its N-terminus and C-terminus. Further, I show that YT is insensitive to negative regulation by the Hippo kinases due to loss of regulatory sites associated with its nuclear export and targeted degradation. Since YT retains the TEAD binding domain at its N-terminus, it suggests that it relies on interaction with TEAD transcription factors to drive its oncogenic transcriptional program. To test this, I utilized biochemical and genetic approaches which show that YT physically interacts with TEAD1, and this interaction is critical for its oncogenic function. Similar observations have previously been described for TC. Importantly, at the time of these studies it was unknown if both gene fusions could promote tumor growth in vivo. Utilizing a mouse xenograft model, I demonstrate that both TC and YT are capable of tumor initiation. What remained to be understood was the functional role for C-terminal fusion partners for both TC and YT. Specifically, I observed that their newly acquired c-termius confer altered DNA binding properties and mediated interactions with chromatin associated factors such as the reader proteins YEATS2 and ZZZ3 of the ATAC histone acetyltransferase complex. Subsequent knockdown of YEATS2 or ZZZ3 in TC/YT expressing cells abrogated the fusion’s ability to promote anchorage independent growth and YEATS2/ZZZ3 knockdown impacted the TC/YT transcriptome resulting in differential regulation of genes such as those involved in the PI3K-AKT and MAPK signaling pathways. Taken together these studies describe a functional role for the c-terminus for TC and YT in the recruitment of chromatin modifying complexes such as the ATAC complex. Importantly, this complex provides a therapeutic target for a sarcoma currently lacking a targeted therapy. The above studies also further demonstrate the importance of chromatin dynamics in determining the final output of TAZ and YAP fusion-based oncogenic transcriptional programs.
In Chapter 3, I explore the crosstalk between PI3K and Hippo signaling pathways in sarcomas. Although activating mutations within the core PI3K signaling pathway are not commonly observed in sarcomas, hyperactivation of the broader PI3K-mTOR signaling network is frequently observed in sarcomas. In vivo, I demonstrated that adenoviral-Cre recombinase mediated knockout of Trp53 and Pten results in the formation of undifferentiated pleomorphic sarcomas (UPS). Further, biochemical and immunohistochemical analysis of primary tumors showed that the PI3K-mTOR pathway is activated as well as TAZ and YAP. To further examine the role of TAZ and YAP downstream of PI3K signaling, we performed a subsequent conditional knockout of both TAZ and YAP, which improved the overall survival of this PI3K activated mouse model of UPS. Additionally, I demonstrated that inhibition of PI3K signaling results in the inhibition of TAZ and YAP in sarcoma cell lines and this is mediated by the LATS1/2 tumor suppressor kinases. Taken together, these studies provide evidence that TAZ and YAP are key oncogenic transcription factors downstream of PI3K signaling in sarcomas and may represent an important signaling axis that mediate resistance to PI3K-mTOR single agent therapies.
Sarcomas are difficult to treat cancers and identifying common therapeutic vulnerabilities may prove to be beneficial when exploring new treatment modalities. The work presented in this thesis describes how two transcriptional co-activators, TAZ and YAP, can become dysregulated in sarcomas. In the vascular sarcoma, EHE, I identify convergent oncogenic functions for the two disease-defining gene fusions, TAZ-CAMTA1 and YAP-TFE3, in which their c-terminal fusion partner interacts with the ATAC histone acetyltransferase complex to augment the basal TAZ and YAP transcriptional program. In addition to gene fusions, I have identified that full-length TAZ and YAP are key downstream effectors of PI3K signaling in sarcomas. Overall, the findings discussed in this thesis support a rationale to explore new ways to effectively target TAZ and YAP in sarcomas.
Details
- Title: Subtitle
- TAZ and YAP dysregulation in sarcomas
- Creators
- Keith Cy Garcia
- Contributors
- Munir R Tanas (Advisor)Kris A DeMali (Committee Member)Rebecca D Dodd (Committee Member)Dawn E Quelle (Committee Member)David J Gordon (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biomedical Science (Cancer Biology)
- Date degree season
- Summer 2022
- DOI
- 10.25820/etd.006506
- Publisher
- University of Iowa
- Number of pages
- xvii, 190 pages
- Copyright
- Copyright 2022 Keith Cy Garcia
- Comment
This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa: https://www.lib.uiowa.edu/sc/contact/.
- Language
- English
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references (pages 176-190).
- Public Abstract (ETD)
Sarcomas are comprised of a diverse group of cancers with over 50 histological types, which are difficult to treat. The mechanisms that drive the development of sarcomas remain to be fully understood. Importantly, due to their extreme heterogeneity, it will prove beneficial to potentially identify shared therapeutic targets to treat this group of cancers which currently lacks effective medical therapies.
The tissues in our body require a coordinated mode of regulation involving controlled cell growth and cell death to maintain proper organ size during development. The Hippo tumor suppressor pathway consists of a series of enzymes that play an important role in the regulation of tissue growth and organ size. Disruption of this signaling pathway can lead to tumor growth. We currently know that the Hippo tumor suppressor pathway plays a critical role in approximately 50-60% of sarcomas. The downstream effectors of the Hippo pathway are two transcriptional co-activators, TAZ (transcriptional coactivator with PDZ-binding motif) and its paralogue YAP (yes associated protein) that are normally inhibited by the Hippo enzymes. In sarcomas and many other forms of cancer, TAZ and YAP are hyperactivated to promote their oncogenic transcriptional program.
In this thesis I explore two ways that TAZ and YAP can become hyperactivated in sarcomas. In the vascular sarcoma called epithelioid hemangioendothelioma (EHE) there are two mutually exclusive gene fusions, TAZ-CAMTA1 (85% of EHE cases) and YAP-TFE3 (15% of EHE cases). These gene fusions are unique such that a portion of the TAZ and YAP proteins are fused to two other proteins, CAMTA1 and TFE3, respectively. I demonstrate that both gene fusions are insensitive to Hippo pathway regulation resulting in their nuclear accumulation and hyperactivation. Additionally, the CAMTA1 and TFE3 components of these gene fusions promote new interactions with epigenetic enzyme complexes such as the Ada-Two-A-Containing (ATAC) complex. These interactions ultimately result in an altered TAZ and YAP transcriptional program that is critical for the transformed phenotype.
In addition to oncogenic TAZ and YAP fusion proteins, I investigate how oncogenic signaling pathways can activate wild-type TAZ and YAP in sarcomas. One such pathway, the phosphoinositide 3-kinase (PI3K) pathway is altered in a wide variety of cancer types including sarcomas. Although progress is being made in the development of novel PI3K inhibitors, targeting the PI3K pathway often shows limited efficacy and is not well tolerated. To gain insight of alternate targets downstream of PI3K signaling, I explored how pharmacological inhibition of this pathway impacts the activity of TAZ and YAP in sarcoma cells. I show that inhibition of PI3K signaling results in the inhibition and degradation of TAZ and YAP in sarcoma cells. Further, removal of TAZ and YAP in mouse tumors that exhibit hyperactivation of PI3K signaling improves their overall survival. Such observations suggest that TAZ and YAP are key oncoproteins downstream of PI3K signaling. These studies identify two oncoproteins that could potentially be targeted in combination with current treatment modalities for sarcomas.
- Academic Unit
- Biomedical Science Program
- Record Identifier
- 9984285347202771
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