Desmoid tumors are rare, locally aggressive neoplasms with unpredictable behavior. Although some tumors are indolent, resolving spontaneously, with tamoxifen, or with anti-inflammatory medications, others are prone to local recurrence, leading to significant morbidity, impaired function and poor quality of life for patients. For patients with advanced, refractory tumors, intensive chemotherapy regimens such as methotrexate/vinblastine or doxorubicin/dacarbazine can produce significant radiographic responses and symptomatic improvement. However these therapies are associated with both short and long-term toxicities. The ability to personalize therapy based on a predictive marker from an individual patient’s tumor would allow early treatment of aggressive tumors with chemotherapy, while sparing patients with indolent tumors or those more likely to respond to milder interventions. Recently, desmoid tumors with particular mutations in CTNNB1, including S45F, have been shown to have a higher risk of recurrence. Additionally, desmoid tumors with S45F mutations were less likely to respond to conservative treatments with imatinib or meloxicam. However, it is unknown whether S45F mutated tumors are more likely to respond to chemotherapy. In this proposal, we will obtain tissue and radiographic imaging for our existing database of desmoid tumor patients, and perform CTNNB1 sequencing to identify specific mutations. Through a collaboration with MD Anderson Cancer Center, our combined datasets will then be analyzed to determine whether the presence of S45F mutations correlates with MRI response to various chemotherapy regimens. The ability to use CTNNB1 mutation status to predict behavior of desmoid tumors and to guide therapeutic decisions would have immediate implications for clinical management of these rare yet often aggressive tumors.
This project aims to provide a fast, semi-high throughput and cheap animal model for identifying and/or characterizing promising drug targets for treating desmoid tumors. In addition the platform allows pre-clinical assessment of novel candidate therapeutic compounds. The project builds on the recent introduction of efficient genome editing methods using TALEN and CRISPR/Cas9, which are creating unique and unmatched opportunities in several research fields, including cancer research. For the first time it is now possible to create functional gene knockouts in a number of model organisms. We have recently generated the first genetic tumor model in the organism Xenopus tropicalis. Because of the external development of the Xenopus embryo and its diploid genome, gene targeting experiments using CRISPR/Cas9 or TALEN are extremely efficient and cheap. Interestingly, when locally targeting the tumor suppressor gene APC we generated tadpoles that rapidly (< 1.5 months) and efficiently (>90%) developed desmoid tumors. This model presents a unique and novel experimental platform that (i) allows the rapid screening and evaluation of genes that contribute to the growth of the tumor, (ii) could serve to assess the clinical relevance of novel drug targets for treating desmoid tumors and (iii) can be used as a preclinical drug screening/assessment. We believe that our model offers a unique experimental platform that can be easily plugged into the research lines of several groups active in the field.
LAY VERSION OF ABSTRACT- Identifying targets for therapy in a novel genetic Xenopus model for desmoid tumor formation
Desmoid tumors (DTs) are rare mesenchymal lesions with a high rate of local recurrence. Their common feature is a deregulated WNT pathway, mainly caused by gain-of-function mutations in exon 3 of the CTNNB1 gene (encoding for beta-catenin), resulting in nuclear accumulation of beta-catenin. Even though it is controversial, several studies have shown that the mutation S45F strongly correlates with increased propensity for desmoid recurrence as compared to T41A mutation. Therefore, it is important to investigate the differences between these 2 genetic alterations in order to identify potential targes for novel molecular therapies. In studies conducted within the premise of our previously funded DTRF seed grant, we were able to establish a desmoid tissue and cell strain repository. Furthermore, we showed that the difference between the CTNNB1 T41A and S45F mutations is not due to differential protein expression levels, since beta-catenin is equally expressed in both mutations. Our gene array analysis showed that pro-apoptotic genes are downregulated and anti-apoptotic genes are upregulated in the cells with the S45F mutation when compared to the T41A mutation. Moreover, we showed that there is no significant induction of apoptosis in the S45F mutated desmoid cell strains when compared to the T41A mutated cells. Interestingly, the impairment of apoptosis appears to be specific to the CTNNB1 S45F mutation and not to desmoid tumors per se. Interim results are not promising, confirming our initial observation. However, these findings are in need of further investigation to identify the molecular mechanisms driving the differences in the induction of apoptosis between the T41A and S45F mutated tumors. Once we understand these differences, these findings may potentially have an impact on patients in the clinic. Finally, we have also started evaluating the effects of agents commonly used for the treatment of DT such as Sorafenib and Imatinib, as potential alternative therapies for patients harboring the beta-catenin S45F mutation. In the second year, we propose to: 1) continue establishing and characterizing human desmoid tumor cell strains, creating a desmoid tumor tissue repository in our new institution as well as to continue trying to establish a desmoid tumor mice model; 2) unravel the molecular mechanism behind the differences in the induction of apoptosis between the CTNNB1 T41A and S45F mutation, and 3) evaluate the efficacy of other therapies for patients harboring the beta-catenin S45F mutation, such as Sorafenib and Imatinib.
Desmoid tumors (DT) are locally invasive soft tissue growths with no directed therapies currently. While two genes (β-catenin and adenomatous polyposis coli) have been found in patients who develop desmoids, it is unclear how these mutations and other downstream mechanisms lead to desmoid tumorigenesis. Extensive research has been explored in the molecular biology of desmoids; however, the use of metabolomics to understand the how the low molecular weight complements of cells, tissues, and biological fluids are perturbed by this highly localized disease. Additionally, the Desmoid Collaboration for a Cure has identified 45 active drugs against primary cell lines. It is unclear how these therapies perturb the metabolome, outside the Wnt and notch pathways. This proposal will use broad spectrum metabolomics to study the tumorigenesis process of fibroblasts to desmoids by investigating paired desmoid and fibroblast cell lines, in addition to unaffected fibroblast cells. Additionally, this proposal will explore the effects of three of the active drugs identified by Collaboration for a Cure on the desmoid and fibroblast cells. It is our hope that this research project will provide knowledge to understand more about the biochemistry of desmoid formation from normal tissue, and how these drugs will help fight desmoid tumors.
LAY VERSION OF ABSTRACT- A metabolomics pilot study on desmoid tumors and novel drug candidates
Aims: To define the natural history and outcomes of patients with desmoid tumors, a rare, locally invasive soft tissue tumor. Methods: Collaborate with Fu Jen University, Taiwan to use a record linkage approach with the Taiwan National Health Insurance Research Data Base (NHIRD) that contains clinical information on 23 million people. Use ICD 10 codes and key words to pull pathology records for review; review 100 records or more to develop an algorithm to ensure specificity; mine the data and develop steps for descriptive epidemiology. Innovation: Data generated from this project will further the understanding of the disease course, impact of available interventions, and the burden of the disease for an individual patient and society as a whole. It will inform the design of future clinical trials for therapeutic interventions in patients with desmoid and could lead to the development of a new treatment for a disease for which there is currently no US Food and Drug Administration (FDA) approved systemic treatment. The pilot project could become the prototype of studying rare diseases using Big Data.
LAY VERSION OF ABSTRACT- Desmoid Tumors for Big Data Linkage
The tumor stroma includes many non-neoplastic cells and the extracellular matrix, making up the microenvironment in which neoplastic cells grow. The non- neoplastic cells are predominantly stromal fibroblasts, sometimes called “tumor associated fibroblasts”. These cells are important for the maintenance and remodeling of the microenvironment, providing the appropriate conditions for neoplastic cell growth and invasion. The role of stromal fibroblasts in promoting tumor invasion has recently been highlighted in a number of cancers such as breast cancer, gastric carcinoma, non-small cell lung carcinoma, and colorectal cancer. This interaction between the stromal fibroblasts and the neoplastic cells can occur indirectly through secreted paracrine factors, or directly by physical cell-cell contact. Desmoid tumors (DTs) are characterized by proliferating and invading fibroblastic cells embedded in depositions of extracellular matrix driven by mutations that activate β-catenin signaling. We hypothesize that desmoid tumor cells interact with the stromal fibroblasts and that this interaction is responsible for maintaining the neoplastic phenotype of tumor cells. To test our hypothesis, we propose to answer the following questions:
Aim 1: How do the neoplastic and stromal populations of desmoid tumors differ?
Aim 2: Do desmoid tumor cells interact with the surrounding stromal cells by paracrine signals?
Aim 3: Do desmoid tumor cells interact with the surrounding stromal cells by direct cell-cell contact?
By elucidating the composition of desmoid tumors, and how the tumor cells interact with the normal stromal cells, we can greatly enhance our knowledge of how they grow. DTs are invasive tumors characterized by abundant extracellular matrix deposition. Therefore, it is very likely that tumor- stromal interactions are important in the way they grow and infiltrate the surrounding tissue. By isolating and characterizing individual cell populations from the tumor mass, we can gain a better understanding of what distinguishes the mutant cells from the normal cells. Our method of separating individual cells will also allow us to study variability between patient samples at the cellular level, without the confounding factor of having a different proportion of normal cells in each tumor sample. Since DTs are known to be heterogeneous, this method is key at understanding the source of this heterogeneity, which can influence treatment decisions. Elucidating what signaling pathways are involved in the process of tumor-stroma interaction will also allow us to potentially identify novel therapeutic targets that we can use to inhibit tumor growth and infiltration. We anticipate that the established single cell-derived clones, and the knowledge gained from this project of desmoid tumor biology, will be the basis for future experiments that build on our knowledge of desmoid tumor heterogeneity and tumor-stromal interactions.
In neoplasia, tumor cells interact with the normal stromal cells, such as the surrounding fibroblasts. This makes studying desmoid tumors difficult as both tumor and stromal cells display a mesenchymal phenotype and there are no well-established markers to distinguish the two populations. To elucidate the composition of desmoid tumors, we isolated and expanded single cells derived from patient desmoid tumor samples. Sequencing of individual clones derived from the same patient sample revealed that desmoid tumors do consist of mutant sub-populations carrying the beta-catenin activating mutation, and normal sub-populations lacking the mutation. To improve our isolation methodology, we performed a high throughput flow cytometry surface antigen screen to study the expression pattern of over 300 surface markers on mutant and non-mutant cells. From this analysis, we identified CD142 as a unique positive marker for the mutant population. Having isolated mutant and non-mutant cell populations, we next looked into the expression of secreted factors that could potentially play a role in cell-cell communication. From this study, we found that CTHRC1 is a factor secreted by the mutant cells. Gain- and loss-of-function experiments showed that CTHRC1 can influence the proliferation rate of desmoid tumor primary cultures. Studying desmoid tumors at the clonal level will enhance our understanding of the intratumoral heterogeneity of these tumors. Identifying unique surface markers allows for the rapid isolation of mutant and non-mutant subpopulations while minimizing cell divisions. In addition to studying tumor-stroma communication, measuring tumor composition may also be useful for ongoing drug screening efforts and as a potential post-treatment readout.
LAY VERSION OF ABSTRACT- Single cell-derived clonal analysis of desmoid tumors to investigate tumor-stroma interactions