Desmoid-type fibromatosis (DF) is a rare connective tissue disease, characterized by the presence of one or more benign non metastasizing myofibroblastic neoplasms with a high local recurrence rate. DF can be diagnosed at any age in both genders but it is more common in young women.

A minority of DF is inheritable and is associated with mutations of the Adenomatous Polyposis Coli (APC) gene, while most of the DF arises sporadically. On the molecular level, 50-80% of the sporadic DF cases have somatic mutations in the codons encoding serine 41 and threonine 45 in exon 3 of the β-catenin gene, CTNNB1. These sites are required for β-catenin phosphorylation and subsequent degradation. Consequently alterations in these sites lead to the Wnt pathway alteration with β-catenin accumulation and its translocation to the nucleus.

Nuclear β-catenin is detected in almost 90% of the desmoid cells and it is considered a positive marker of DF cells once malignant cell transformation is excluded. In addition to this marker, we recently demonstrated that GSK-3β, also involved in Wnt pathway regulation, translocates in the nucleus of DF cells as well, colocalizing and interacting with β-catenin. This event occurs regardless of the presence of CTNNB1 gene mutations suggesting that the β-catenin/GSK-3β interaction and the nuclear translocation can be unrelated to the genetic alterations of CTNNB1. This led us to formulate the hypothesis that the alteration Wnt/β-catenin signaling pathway in desmoid tumor is modulated by the interaction with microenvironmental factors.

The aim of this project is the identification and the characterization of the microenvironment factors responsible for the proliferation and aggressiveness of DF cells. Since the deregulation of the inflammatory process can lead to aberrant fibroblast activation and accumulation of ECM proteins with subsequent tissue fibrosis that can evolve in fibrotic disease and tumor development, we will focus our attention on the inflammatory and growth factors and their interaction with the Wnt/β-catenin pathway. For this purpose cells derived from desmoid tumor and control cell samples will be incubated with different cytokines, growth factors and hormones. Their effect on the growth rate and aggressiveness will be evaluated by immunofluorescence using specific antibodies for cell cycle markers, for the receptors of the microenvironmental factors, for the proteinases degrading the extracellular matrix, for cysteine proteases and for the markers of the basal membrane, lysosomes and vesicles.

We will use a custom Ion AmpliSeq RNA panel to analyze the gene expression of DF cells under treatment, in particular focusing on genes of the Wnt pathway and of the other correlated pathways. Modulating the microenvironment we expect to identify potential factors that promote the DF tumor growth and aggressiveness. This is a critical milestone in defining the potential targets for a therapy aimed at reducing the proliferation, local aggressiveness and recurrence rate of DF tumors.

This study is part of a long term multidisciplinary research program that is ongoing since 2011 and it aims to improve the knowledge of the Desmoid-type fibromatosis and to identify a viable treatment. To date the study has led to the identification of a second potential positive marker of desmoid tumor cells, the nuclear translocation of GSK-3β.

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Although desmoid clinical biology has been well characterized, the molecular biology of those tumors tumor remains poorly understood, perhaps because of the global lack of needed research bioresources such as desmoid cells and human desmoid tissue banks needed for the high throughput research necessary to identify mutated genes and abnormal proteins driving desmoid tumor inception, progression, invasion, treatment resistance, and recurrence. In studies conducted within the premise of our DTRF- funded seed grants we were able to establish a large desmoid tissue and cell strain repository in our former lab and have identified WKDW ȕ-catenin mutations are highly prevalent in sporadic desmoid tumors and that a specific mutation, S45F possibly significantly correlates with dismal patient outcome. Moreover, Hamada et al. have shown that CTNNB1 S45F mutation predicts poor efficacy of meloxicam treatment for DT. Those interesting initial observations led further investigation by our group.

We performed a gene array which we included 44 desmoid tumor tissues (14 tumors harboring the 45F mutation, 16 tumors with a 41A mutation and 14 wild type tumors), 14 corresponding normal tissues and 14 desmoid cell strains (8 cells strains with a 41A mutation and 6 cell strains with a 45F mutation). Our initial results showed that the mutation T41A was enriched with pro apoptosis gene and that negative apoptosis regulator genes were upregulated in the mutation S45F. In the current application we suggest to continue our studies and propose a two-pronged research design consisting of distinct yet interrelated components that will address current knowledge deficits. Our studies will focus on reestablishing and characterizing new human desmoid tumor cell strains, recreating a desmoid tumor tissue repository as well as establishing a desmoid tumor mice model in our new lab at the Ohio State University. Furthermore, understanding that the deregulation of the beta-catenin pathway is potentially a significant contributor to tumorigenesis and progression and the limitation of direct therapeutic targeting of E-catenin we would attempt to identify potential targets that could reactivate the apoptosis which may be related to the aggressive behavior of the desmoid tumors harboring the CTNNB1 S45F mutation. Identifying genes and their cognate proteins whose alteration would explain the aggressive behavior of some desmoid tumors might provide future targets for novel molecular therapies relevant to patients burdened by this devastating disease.

Desmoid tumors (DTs) are mesenchymal tumors that occur sporadically or in the context familial adenomatous polyposis (FAP). Although rare in the general population, DTs are a leading cause of death in FAP patients. There is no effective cure for patients with unresectable disease, and little is known about the molecular biology driving desmoid tumorigenesis. Our laboratory seeks to characterize the biological requirements necessary for DT formation and identify targeted therapies for this disease. Currently, we are investigating the role of hyaluronic acid (HA) in driving desmoid proliferation and have preliminary evidence that HA is an abundant constituent of DTs.

Based on our novel observations, we hypothesize that hyaluronic acid plays a critical role in desmoid tumorigenesis and provides a novel target for therapy. Furthermore, we propose that HA promotes DT progression through crosstalk with inflammatory components of the tumor microenvironment such as mesenchymal stem cells (MSCs). We will test this hypothesis through two specific and independent aims:

Aim 1: To evaluate the anti-tumorigenic effects of hyaluronic acid inhibition in human desmoid-derived cells. Aim 2: To evaluate the anti-tumorigenic effects of hyaluronic acid inhibition in the Apc1638N mouse and xenograft models. Our findings will 1) address major gaps in our current understanding of DT pathophysiology, 2) define the role that HA plays in driving desmoid tumorigenesis, and 3) identify novel targets for therapy. As a clinical referral center for patients with DTs, we are uniquely situated to carry out this proposal given our access to rare patient tumors that will sustain our future work and allow us to expeditiously translate benchside discoveries to clinical trials.

Desmoid tumors (DT) are locally invasive lesions that are difficult to treat using conventional therapies. While there is some success with chemotherapy in people who have recurrence of their tumors after surgery and/or radiation, much of the time the results from chemotherapy are temporary, and regrowth of tumor is seen. One approach to treatment is to develop a combination of medications that target different aspects of tumor growth, but have few side effects. Since desmoids are locally invasive but do not metastasize to other locations in the body, it is not necessary to wipe out every tumor cell, but just to get the tumor to shrink. We assembled a consortium of researchers from different backgrounds, and with complementary skills, whose ultimate goal is to develop a treatment to therapeutically target tumor cells in desmoid patients without causing serious side effects.

To achieve this goal, over the past year, we screened drugs in the laboratory on cell lines from desmoid tumor patients to see which agents specifically target tumor cell viability, and are in the process of testing the drugs in cell lines. Over the remaining course of the proposal we plan to: (1) Complete the testing of drugs identified in the screen using cell lines from a variety of desmoid tumors to determine the mechanism by which they inhibit cell viability. (2) Test drugs that are effective in cell lines in a mouse model that is engineered to develop desmoids. (3) The agents identified in the screen will then be analyzed for the ability to use them in combination, and these combinations will be tested in both desmoid cell cultures and in genetically modified mice.

In the past year we screened eight cell lines using the Maybridge HitFinderTM Collection; DIVERSetTM Collection (Chembridge); LOPAC (Sigma); BIOMOL collection; Prestwick collection (Prestwick Chemical); Seminatural compounds (AnalytiCon Discovery); and the NIH Clinical Collection (BioFocusDPI). This screening identified 45 compounds that inhibited cell proliferation in desmoid tumors by greater than 50% while not affecting normal fibroblast proliferation, and that also are either in use in patients or that have the potential to be rapidly developed for patient care.

We are currently testing these drugs using a large collection of desmoid tumor cell cultures of different genetic etiologies, and based on this, will test them in mice that develop desmoid tumors. Drugs that inhibit desmoid tumor growth in both cells and mice will then be studied using pharmacokinetic analysis, to identify drugs that work in an additive fashion when used in combination, but will still have an acceptable safety profile. We will then test the drugs already approved for use in patients in a multi drug regimen as informed by pharmacokinetic data. Then these combinations will be tested in multiple human desmoid tumor cultures and in genetically modified mouse models.

New candidate drugs will also be analyzed to identify their underlying biologic mechanism, to identify novel pathways that are activated by beta-catenin in desmoid tumors. These data will be used to prioritize new research into the biology of how beta-catenin causes desmoid tumors. This approach should rapidly identify a multi-drug combination that can then safely be tested for efficacy in patients. In addition, it will identify new pathways activated by beta-catenin and new agents not yet in use for patient care that target desmoid tumors, both of which can be developed in future work which might ultimately identify even more effective therapeutic approaches.