Browsing by Author "Alman, Benjamin A"
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Item Open Access CD142 Identifies Neoplastic Desmoid Tumor Cells, Uncovering Interactions Between Neoplastic and Stromal Cells That Drive Proliferation.(Cancer research communications, 2023-04) Al-Jazrawe, Mushriq; Xu, Steven; Poon, Raymond; Wei, Qingxia; Przybyl, Joanna; Varma, Sushama; van de Rijn, Matt; Alman, Benjamin AThe 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.Item Open Access Loss of the Chromatin Remodeler, ATRX, Promotes Aggressive Features of Osteosarcoma and Activates NF-κB Signaling and Integrin Receptor Binding(2021) Bartholf DeWitt, SuzanneOsteosarcoma (OS) is a lethal disease with few known targeted therapies. Recent studies of OS have begun to recognize the relatively common mutations in and subsequent loss-of-function of the gene, ATRX, however the biological impact of the loss of this gene on tumor biology is not well-understood. Thus, exploring the role of ATRX expression loss in OS tumorigenesis may provide a better understanding of this disease as well as guide us to potential new and much needed targeted therapies based on expression of this gene. Here we investigated how loss of function of the chromatin remodeler, ATRX, impacts OS biology and contributes to aggressive disease phenotypes, and we identified the cellular signaling pathways driving these altered phenotypes. In order to explore the role of ATRX loss in mouse OS, we used a previously established Osterix-Cre driven genetically engineered mouse model of OS to examine tumor initiation in mice with loss of Rb and p53 compared to loss of Rb, p53, and ATRX. In this mouse model, ATRX loss correlated with increased tumor initiation relative to wildtype ATRX expression. To investigate how ATRX loss impacts human OS cellular phenotypes of aggression, we stably transduced the human 143B OS cell line with a non-silencing shRNA or one of two independent shRNA constructs for ATRX knockdown, and ATRX knockout 143B and MG63 cell lines were generated using CRISPR-Cas9. These cell lines were then manipulated both in vitro and in vivo to characterize the resulting phenotypic changes of tumor growth, migration, invasion and metastasis in human OS. ATRX knockdown or knockout in the 143B human OS cell line enhanced growth and local invasion of established xenograft tumors. These knockdown and knockout cells displayed significantly greater migration and invasion than the wildtype cells in both scratch wound and transwell migration and invasion assays. An orthotopic mouse model showed increased lung metastasis in mice injected with the 143B ATRX knockout cell line compared to wildtype. In order to further elucidate the cellular mechanisms that correspond with the phenotypic changes found with loss of ATRX expression, RNA-Seq and ATAC-Seq were performed on the 143B ATRX shRNA knockdown and non-silenced control cells. In the sequencing results, several NF-κB-associated pathways were most significantly upregulated (based on normalized enrichment scores) and several extracellular matrix (ECM) pathways were most significantly downregulated, supporting a role for ATRX in matrix remodeling and invasion. NF-κB ELISA assays were used to further validate the sequencing data, and a chromatin binding motif analysis identified a common overlap with ETS transcription factor motifs. Wildtype or CRISPR-Cas9 knockout MG63 cells were screened with 2,100 bioactive small molecule inhibitors to identify drugs for which ATRX loss of function led to increased drug efficacy, and there was significant sensitization of the ATRX knockout to an integrin inhibitor, SB273005. An in vivo study in an ATRX-null U2OS xenograft mouse model showed reduced tumor growth with this integrin inhibitor compared to vehicle-treated mice. Additionally, this integrin inhibitor reversed the increased phenotypes of aggression seen with ATRX loss in vitro, including migration and invasion, and this drug partially reversed the nuclear upregulation of the NF-κB transcription factors. Thus, our studies show that functional ATRX plays an important tumor suppression role in OS, and decreased ATRX expression is associated with more aggressive cellular phenotypes, including increased growth, migration, invasion and metastasis. Here we show a correlation between ATRX deficiency, these aggressive phenotypes, activation of NF-κB signaling, increased integrin αvβ3 expression, altered ECM remodeling, and ETS transcription factor binding. However, ATRX-deficient cells display substantially increased sensitivity to integrin signaling inhibition. The relationship of ATRX expression with integrin-binding, NF-κB activation, and ETS transcription factor binding has not been noted in previous studies, but may impact other known diseases with ATRX loss, including other cancers and ATR-X mental retardation syndrome. Future studies are needed to explore integrin inhibition as a potential new targeted therapy for ATRX-deficient OS.
Item Open Access Macrophage cells secrete factors including LRP1 that orchestrate the rejuvenation of bone repair in mice.(Nature communications, 2018-12-05) Vi, Linda; Baht, Gurpreet S; Soderblom, Erik J; Whetstone, Heather; Wei, Qingxia; Furman, Bridgette; Puviindran, Vijitha; Nadesan, Puviindran; Foster, Matthew; Poon, Raymond; White, James P; Yahara, Yasuhito; Ng, Adeline; Barrientos, Tomasa; Grynpas, Marc; Mosely, M Arthur; Alman, Benjamin AThe pace of repair declines with age and, while exposure to a young circulation can rejuvenate fracture repair, the cell types and factors responsible for rejuvenation are unknown. Here we report that young macrophage cells produce factors that promote osteoblast differentiation of old bone marrow stromal cells. Heterochronic parabiosis exploiting young mice in which macrophages can be depleted and fractionated bone marrow transplantation experiments show that young macrophages rejuvenate fracture repair, and old macrophage cells slow healing in young mice. Proteomic analysis of the secretomes identify differential proteins secreted between old and young macrophages, such as low-density lipoprotein receptor-related protein 1 (Lrp1). Lrp1 is produced by young cells, and depleting Lrp1 abrogates the ability to rejuvenate fracture repair, while treating old mice with recombinant Lrp1 improves fracture healing. Macrophages and proteins they secrete orchestrate the fracture repair process, and young cells produce proteins that rejuvenate fracture repair in mice.Item Open Access Regulation of Cartilage Tumors by Mutations in Isocitrate Dehydrogenases(2021) Zhang, HongyuanEnchondroma and chondrosarcoma are common benign and malignant cartilaginous neoplasms. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are present in the majority of these tumors. Mutant IDH enzymes gain a neomorphic function of producing D-2-hydroxyglutarate (D-2HG) from ?-ketoglutarate. Expression of a mutant Idh1 gene is sufficient for enchondroma initiation but inhibiting mutant IDH enzymes did not cause a consistent change in the tumorigenic properties of chondrosarcomas. It is unclear how mutations of isocitrate dehydrogenases regulate cartilage tumors from initiation to cancer progression and maintenance. I hypothesize that mutations in IDH enzymes could regulate cartilage tumors through changes in gene expression and cellular metabolism. To address these questions, I examined the transcriptional regulation and metabolic regulations of mutant isocitrate dehydrogenases in chondrocytes and chondrosarcomas and identified cholesterol biosynthesis and glutamine metabolism as two key pathways dictating tumor behavior.
To understand the transcriptional regulation of IDH1 mutation in cartilage tumors, I performed RNA-sequencing analysis in chondrocytes from Col2a1Cre;Idh1LSL/+ mutant animals and their littermate wildtype controls and identified that cholesterol biosynthesis pathway was upregulated. Genetic inhibition of cholesterol biosynthesis in an enchondroma mouse model and pharmacological inhibition of cholesterol biosynthesis in human patient chondrosarcoma samples suppressed tumor development in vivo. Taken together, these data suggest that intracellular cholesterol synthesis is a potential therapeutic target for enchondromas and chondrosarcomas.
From a metabolic perspective, I found that chondrocytes and chondrosarcomas with mutations in IDH1/2 genes had enhanced glutamine utilization for downstream metabolism. Using genetic and pharmacological approaches, I demonstrated that glutaminase-mediated glutamine metabolism played distinct roles in enchondromas and chondrosarcomas with IDH1/2 mutations. Genetic ablation of glutaminase in chondrocytes with Idh1 mutation increased the number and size of enchondroma-like lesions. Pharmacological inhibition of glutaminase led to decreased tumor weight of chondrosarcoma xenograft. During enchondroma development, glutamine-derived -ketoglutarate plays important roles in regulating chondrocyte differentiation and proliferation. In chondrosarcoma, glutamine-derived non-essential amino acids are important in preventing cell apoptosis.
In summary, findings in this dissertation described transcriptional and metabolic regulations by mutations in isocitrate dehydrogenases in cartilage tumors enchondroma and chondrosarcoma and provide novel insights for developing therapies for these diseases.