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The interaction between neoplastic and stromal cells within a tumor mass plays an important role in cancer biology. However, it is challenging to distinguish between tumor and stromal cells in mesenchymal tumors because lineage-specific cell surface markers typically used in other cancers do not distinguish between the different cell subpopulations. Desmoid tumors consist of mesenchymal fibroblast-like cells driven by mutations stabilizing beta-catenin. Here we aimed to identify surface markers that can distinguish mutant cells from stromal cells to study tumor-stroma interactions. We analyzed colonies derived from single cells from human desmoid tumors using a high-throughput surface antigen screen, to characterize the mutant and nonmutant cells. We found that CD142 is highly expressed by the mutant cell populations and correlates with beta-catenin activity. CD142-based cell sorting isolated the mutant population from heterogeneous samples, including one where no mutation was previously detected by traditional Sanger sequencing. We then studied the secretome of mutant and nonmutant fibroblastic cells. PTX3 is one stroma-derived secreted factor that increases mutant cell proliferation via STAT6 activation. These data demonstrate a sensitive method to quantify and distinguish neoplastic from stromal cells in mesenchymal tumors. It identifies proteins secreted by nonmutant cells that regulate mutant cell proliferation that could be therapeutically., Significance: Distinguishing between neoplastic (tumor) and non-neoplastic (stromal) cells within mesenchymal tumors is particularly challenging, because lineage-specific cell surface markers typically used in other cancers do not differentiate between the different cell subpopulations. Here, we developed a strategy combining clonal expansion with surface proteome profiling to identify markers for quantifying and isolating mutant and nonmutant cell subpopulations in desmoid tumors, and to study their interactions via soluble factors., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Introduction: Desmoid-type fibromatosis (DTF) is a rare, soft tissue tumour. Sorafenib, a multikinase inhibitor, has demonstrated antitumour efficacy in DTF patients. Little is known about the underlying molecular mechanisms, which are crucial to know to further optimize systemic treatments. Here we investigated the molecular effects of sorafenib exposure on DTF and stromal cells, with an emphasis on cell death mechanisms., Material and Methods: DTF primary cell cultures, with known CTNNB1 status, and primary stromal cell cultures, derived from DTF tissue, were exposed to clinically relevant concentrations of sorafenib in the presence or absence of inhibitors of ferroptosis, apoptosis and autophagy. Cell viability was determined after 24 and 48 h using MTT assays. Annexin V/PI staining, lipid peroxidation analysis and immunoblotting were performed to assess apoptosis, ferroptosis and autophagy., Results: Exposure to sorafenib caused a significant, concentration- and time-dependent decrease in cell viability in all primary DTF and stromal cell cultures. Inhibitors of ferroptosis and apoptosis protected against sorafenib-mediated cytotoxicity implicating that both cell death mechanisms are activated. Annexin V/PI stainings and lipid peroxidation analyses confirmed induction of apoptosis and ferroptosis, respectively. Autophagy inhibition enhanced the cytotoxic effect of sorafenib and led to a stronger induction of apoptosis and ferroptosis., Conclusion: This study identified ferroptosis and apoptosis as mechanisms for the sorafenib induced cell death in DTF cells as well as stromal cells. Furthermore, autophagy inhibition enhanced the cytotoxic effects of sorafenib. Knowledge of the mechanisms by which sorafenib affects DTF at a cellular level may help to optimize its clinical efficacy and mitigate toxic effects., Competing Interests: Declaration of competing interest None., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cancer precision medicine implies identification of tumor-specific vulnerabilities associated with defined oncogenic pathways. Desmoid tumors are soft-tissue neoplasms strictly driven by Wnt signaling network hyperactivation. Despite this clearly defined genetic etiology and the strict and unique implication of the Wnt/β-catenin pathway, no specific molecular targets for these tumors have been identified. To address this caveat, we developed fast, efficient, and penetrant genetic Xenopus tropicalis desmoid tumor models to identify and characterize drug targets. We used multiplexed CRISPR/Cas9 genome editing in these models to simultaneously target a tumor suppressor gene ( apc ) and candidate dependency genes. Our methodology CRISPR/Cas9 selection-mediated identification of dependencies (CRISPR-SID) uses calculated deviations between experimentally observed gene editing outcomes and deep-learning-predicted double-strand break repair patterns to identify genes under negative selection during tumorigenesis. This revealed EZH2 and SUZ12 , both encoding polycomb repressive complex 2 components, and the transcription factor CREB3L1 as genetic dependencies for desmoid tumors. In vivo EZH2 inhibition by Tazemetostat induced partial regression of established autochthonous tumors. In vitro models of patient desmoid tumor cells revealed a direct effect of Tazemetostat on Wnt pathway activity. CRISPR-SID represents a potent approach for in vivo mapping of tumor vulnerabilities and drug target identification., Competing Interests: The authors declare no competing interest.

Enchondroma and chondrosarcoma are the most common benign and malignant cartilaginous neoplasms. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are present in the majority of these tumors. We performed RNA-seq analysis on chondrocytes from Col2a1Cre;Idh1LSL/+ animals and found that genes implied in cholesterol synthesis pathway were significantly upregulated in the mutant chondrocytes. We examined the phenotypic effect of inhibiting intracellular cholesterol biosynthesis on enchondroma formation by conditionally deleting SCAP (sterol regulatory element-binding protein cleavage-activating protein), a protein activating intracellular cholesterol synthesis, in IDH1 mutant mice. We found fewer enchondromas in animals lacking SCAP. Furthermore, in chondrosarcomas, pharmacological inhibition of intracellular cholesterol synthesis significantly reduced chondrosarcoma cell viability in vitro and suppressed tumor growth in vivo. Taken together, these data suggest that intracellular cholesterol synthesis is a potential therapeutic target for enchondromas and chondrosarcomas.

Desmoid tumors (aggressive fibromatosis) are locally invasive soft tissue tumors that lack the ability to metastasize. There are no directed therapies or standard treatment plan, and chemotherapeutics, radiation, and surgery often have temporary effects. The majority of desmoid tumors are related to T41A and S45F mutations of the beta-catenin encoding gene (CTNNB1). Using broad spectrum metabolomics, differences were investigated between paired normal fibroblast and desmoid tumor cells from affected patients. There were differences identified, also, in the metabolomics profiles associated with the two beta-catenin mutations, T41A and S45F. Ongoing drug screening has identified currently available compounds which inhibited desmoid tumor cellular growth by more than 50% but did not affect normal fibroblast proliferation. Two drugs were investigated in this study, and Dasatinib and FAK Inhibitor 14 treatments resulted in unique metabolomics profiles for the normal fibroblast and desmoid tumor cells, in addition to the T41A and S45F. The biochemical pathways that differentiated the cell lines were aminoacyl-tRNA biosynthesis in mitochondria and cytoplasm and signal transduction amino acid-dependent mTORC1 activation. This study provides preliminary understanding of the metabolic differences of paired normal and desmoid tumors cells, their response to desmoid tumor therapeutics, and new pathways to target for therapy.

Both the WNT/β-catenin and hedgehog signaling pathways are important in the regulation of limb development, chondrocyte differentiation, and degeneration of articular cartilage in osteoarthritis (OA). It is not clear how these signaling pathways interact in interzone cell differentiation and synovial joint morphogenesis. Here, we determined that constitutive activation of hedgehog signaling specifically within interzone cells induces joint morphological changes by selectively inhibiting β-catenin-induced Fgf18 expression. Stabilization of β-catenin or treatment with FGF18 rescued hedgehog-induced phenotypes. Hedgehog signaling induced expression of a dominant negative isoform of TCF7L2 (dnTCF7L2) in interzone progeny, which may account for the selective regulation of β-catenin target genes observed. Knockdown of TCF7L2 isoforms in mouse chondrocytes rescued hedgehog signaling-induced Fgf18 downregulation, while overexpression of the human dnTCF7L2 orthologue (dnTCF4) in human chondrocytes promoted the expression of catabolic enzymes associated with OA. Similarly, expression of dnTCF4 in human chondrocytes positively correlated with the aggrecanase ADAMTS4. Consistent with our developmental findings, activation of β-catenin also attenuated hedgehog-induced or surgically induced articular cartilage degeneration in mouse models of OA. Thus, our results demonstrate that hedgehog inhibits selective β-catenin target gene expression to direct interzone progeny fates and articular cartilage development and disease. Moreover, agents that increase β-catenin activity have the potential to therapeutically attenuate articular cartilage degeneration as part of OA.

Objective: With no effective therapies to attenuate cartilage degeneration in osteoarthritis (OA), the result is pain and disability. Activation of hedgehog (HH) signaling causes changes related to the progression of OA, with higher levels of Gli-mediated transcriptional activation associated with increased disease severity. To elucidate the mechanism through which this occurs, this study sought to identify genes regulated by HH signaling in human OA chondrocytes., Methods: Using human OA cartilage samples, microarray analyses were performed to detect changes in gene expression when the HH pathway was modulated. Results were analyzed for differentially expressed genes, grouped into functional networks, and validated in independent samples. To investigate the effects of chondrocyte-specific sterol accumulation, we generated mice lacking Insig1 and Insig2, which are major negative regulators of cholesterol homeostasis, under Col2a1 regulatory elements., Results: HH signaling was found to regulate genes that govern cholesterol homeostasis, and this led to alterations in cholesterol accumulation in chondrocytes. A higher level of Gli-mediated transcription resulted in accumulation of intracellular cholesterol. In genetically modified mice, chondrocyte-specific cholesterol accumulation was associated with an OA phenotype. Reducing cholesterol accumulation attenuated the severity of OA in mice in vivo and decreased the expression of proteases in human OA cartilage in vitro., Conclusion: HH signaling regulates cholesterol homeostasis in chondrocytes, and intracellular cholesterol accumulation contributes to the severity of OA. Our findings have therapeutic implications, since reduction of HH signaling reversed cholesterol accumulation and statin treatment attenuated cartilage degeneration., (© 2016 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.)

Desmoid tumors, or aggressive fibromatosis, are rare, locally infiltrative neoplasms caused by mutations that activate β-catenin. Although these tumors do not metastasize, they are difficult to manage due to variability in tumor presentation and behavior. A variety of treatment options exist, including surgery, radiotherapy, chemotherapy, hormone therapy, isolated limb perfusion, cryoablation and tyrosine kinase inhibitors. Treatment-induced morbidity and poor local control rates, combined with spontaneous stabilization of some desmoid tumors, have allowed watchful waiting to recently emerge as a front-line management option. This has emphasized the need to better understand tumor behavior in order to differentiate between tumors that may stabilize and those that may progress. Here, we review the most recent findings in desmoid tumor biology and treatment options for this enigmatic disease.

Desmoid tumor (also called aggressive fibromatosis) is a lesion of mesenchymal origin that can occur as a sporadic tumor or a manifestation of the preneoplastic syndrome, familial adenomatous polyposis caused by a mutation in adenomatous polyposis coli (APC). This tumor type is characterized by the stabilization of β-catenin and activation of Tcf-mediated transcription. Cell transplantation data suggest that desmoid tumors are derived from mesenchymal progenitor cells (MSCs). As such, modulating cell signaling pathways that regulate MSC differentiation or proliferation, such as hedgehog (Hh) signaling, could alter the tumor phenotype. Here, we found that Hh signaling is activated in human and murine desmoid tumors. Inhibiting Hh signaling in human cell cultures decreased cell proliferation and β-catenin protein levels. Apc(+)/Apc(1638N) mice, which develop desmoid tumors, develop smaller and fewer tumors when Hh signaling was inhibited either genetically (by crossing Apc(+)/Apc(1638N) mice with mice lacking one copy of a Hh-activated transcription factor, Gli2 (+/-) mice) or using a pharmacologic inhibitor. Both in mice and in human tumor cell cultures, β-catenin and Hh-mediated signaling positively regulate each other's activity. These data show that targeting a pathway that regulates MSC differentiation influences desmoid tumor behavior, providing functional evidence supporting the notion that these tumors are derived from mesenchymal progenitors. It also suggests Hh blockade as a therapeutic approach for this tumor type.