Ewing sarcoma (ES) involves a tumor-specific chromosomal translocation that produces the EWS-FLI1 protein, which is required for the growth of ES cells both in vitro and in vivo. However, an ...EWS-FLI1-driven transgenic mouse model is not currently available. Here, we present data from six independent laboratories seeking an alternative approach to express EWS-FLI1 in different murine tissues. We used the Runx2, Col1a2.3, Col1a3.6, Prx1, CAG, Nse, NEFL, Dermo1, P0, Sox9 and Osterix promoters to target EWS-FLI1 or Cre expression. Additional approaches included the induction of an endogenous chromosomal translocation, in utero knock-in, and the injection of Cre-expressing adenovirus to induce EWS-FLI1 expression locally in multiple lineages. Most models resulted in embryonic lethality or developmental defects. EWS-FLI1-induced apoptosis, promoter leakiness, the lack of potential cofactors, and the difficulty of expressing EWS-FLI1 in specific sites were considered the primary reasons for the failed attempts to create a transgenic mouse model of ES.
Abstract
Ewing sarcoma (EwS) is driven by the EWS-FLI1 (EF) oncoprotein and is assumed to originate from mesenchymal progenitors during bone development. Attempts to generate a EwS genetic mouse ...model by targeting EF expression to the mesenchymal lineage so far failed to result in tumorigenesis. Here, we investigated the influence of bone niche-derived factors on EF dependent transformation. We focused on IGF1 which is highly enriched in the bone during puberty when EwS incidence peaks, and which was previously proposed to play an essential role in EwS pathogenesis. We restricted EF expression to mesenchymal stem cells during endochondral bone formation by crossing an EF knock-in mouse with a mouse expressing Prx1-driven Cre recombinase. The resulting EFprx1 progeny died after birth as consequence of severe skeletal malformations resulting from differentiation arrest of early chondrocytes. In vitro, these embryonic EF transgenic chondrocyte-like mesenchymal stem cells (EFprx1 MSCLC) are immortal, but largely failed to form colonies in soft-agar as functional surrogate of full malignant transformation. In contrast, when treated with IGF1 for 4 weeks, a significant increase in soft-agar colony formation was observed, which was dependent on sustained EF expression. Strikingly, cells from these colonies maintained their transformed phenotype in absence of further IGF1 treatment and gave rise to aggressively growing tumors upon xenotransplantation in mice. Analysis of differentially expressed genes from these stably IGF1-reprogrammed, fully transformed EFprx1 MSCLC showed a significant enrichment for the EwS specific EF transcriptional signature with upregulation of Foxm1 and cell cycle-related pathways, and down-regulation of apoptotic gene sets. Cluster analysis of open chromatin regions in EF immortalized versus fully transformed and wildtype cells revealed five distinct chromatin modules. Of these, module 2 comprises genomic regions that open up exclusively in IGF1 reprogrammed EF expressing cells. Among associated genes, Yap1 was most highly induced in IGF1 reprogrammed EFprx1 MSCLC. Consistent with this finding, RNA-seq revealed enrichment of a Yap1 transcriptional signature in IGF1 reprogrammed versus parental EF expressing cells. In addition, chromatin module 4, which comprises regions open in parental and IGF1 reprogrammed EFprx1 MSCLC but not in wildtype MSC, is highly enriched in binding motifs for TEAD transcription factors, the functional effectors of YAP1 signaling. We therefore hypothesize that YAP1 induction by IGF1 reprogramming synergizes with EF in the activation of module 4 genes. Strikingly, modules 2 and 4 comprise hallmark genes of stemness and self-renewal which also characterize human EwS, including Bcl11b, Prkcb, Sox2, Ccnd1, and Klf5, as well as the top EwS dependency gene Igf2bp1. Our data suggest that transient IGF1 exposure in the bone microenvironment during puberty may be required for EF driven EwS tumor initiation, and we identify a central role of YAP1 in the early phases of EwS pathogenesis.
Citation Format: Heinrich Kovar, Rahil Noorizadeh, Florian Halbritter, Utkarsh Kapoor, Wolfgang Mikulits, Richard Moriggl, Barbara Sax. YAP1 is a key regulator of EWS-FLI1-dependent malignant transformation upon IGF1 mediated reprogramming of bone mesenchymal stem cell-like cells abstract. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR003.
Abstract
Ewing sarcoma (EwS), an aggressive bone and soft tissue cancer predominantly occurring during adolescence, is driven by the chimeric EWS-FLI1 oncogene. It is assumed that a permissive ...cellular context or potentially additional complementing mutations are necessary for the EWS-FLI1 fusion to exert its full oncogenic effect. Prior efforts to generate genetic EwS mouse models failed because of high EWS-FLI1 induced apoptosis when expressed in various candidate progenitor cell types. In our study, we restricted EWS-FLI1 expression to mesenchymal stem cells during endochondral bone formation by crossing a mouse containing EWS-FLI1 knocked into the Rosa26 locus with a mouse expressing Prx1-driven Cre recombinase. However, the resulting EFprx1 progeny died after birth as functional consequence of severe skeletal malformations resulting from differentiation arrest of early chondrocytes. In vitro, these embryonic EWS-FLI1 transgenic chondrocyte-like mesenchymal stem cells (EFprx1 MSCLC) are immortal, but not fully transformed. However, we observed elevated levels of cleaved PARP-1, a surrogate marker of apoptosis. This apparent increased cellular turnover was paralleled by down-regulation of anti-apoptotic Bcl2a family genes as identified by RNA-seq analysis of EFprx1 MSCLC. The peak incidence of human EwS occurs during puberty, a developmental period in which several growth promoting hormones are highly enriched in the bone microenvironment. Among them is IGF1, which is known for its anti-apoptotic activity through upregulation of Bcl2 family genes. As previous studies implied IGF1 signaling essential in EwS pathogenesis, we speculated that IGF1 treatment may aid in the transformation process of EFprx1 MSCLC. In fact, a significant increase in soft-agar colony formation was observed upon IGF1 treatment for 4 weeks. To test whether IGF1 had irreversibly reprogrammed EFPrx1 MSCLC, we picked colonies grown in presence of IGF1, expanded and re-assayed them after 5 weeks in absence of the hormone. Strikingly, EFprx1 MSCLC retained their transformed phenotype not only in the soft-agar assay but also upon xenotransplantation in mice. Gene set enrichment analysis of upregulated genes from these stably hormone-reprogramed fully transformed EFprx1 MSCLC showed a significant enrichment for the EWS-FLI1 specific transcriptional signature and cell cycle-related pathways, while apoptotic gene sets were downregulated. Our data suggest that transient IGF1 exposure in the bone microenvironment may be required for EWS-FLI1 driven transformation. We propose that EwS formation could be influenced by hormonal changes during puberty. Based on these findings, we postulate that correct developmental timing of EWS-FLI1 expression may play a pivotal role for the generation of an EwS mouse model.
Citation Format: Rahil Noorizadeh, Florian Halbritter, Barbara Sax, Wolfgang Mikulits, Richard Moriggl, Heinrich Kovar. Transient IGF1 exposure stably reprograms EWS-FLI1 immortalized embryonal limb-derived mesenchymal stem cell-like cells to full transformation abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1669.
Futher, a majority of these purchases are impulse buys "Consumers want to focus their daily diet on the best-tasting products, leading to a shift from center store packaged goods to in-store bakery ...items that are fresher andmore indulgent," says Brant Cash, managing director at New York-based global investment bank Harris Williams. Lakeland, Fla.-based Publix Super Markets recently debuted Italian-inspired Tutto Pugliese bread, made exclusively from durum wheat semolina flour and certified sourdough flour,which are typically used to make pasta. Since many baked goods, such as cookies and brownies, travel very well through delivery channels, Wisloski suggests that supermarkets can take a cue from restaurants that are capturing impulse sales of baked goods with "add to order" reminders on their platforms. At Dom's Kitchen & Market location in Chicago's Lincoln Park neighborhood, customers can choose from a variety of Bond Roman-style pizzas, including Potato & Mozzarella, Ricotta Zucchini, Sopressata, Spicy Eggplant, Sun Dried Tomato Arugula, and Mushroom Rosa.
...retailers are investing heavily in foodservice. ...82% of U.S. retailers are increasing space for fresh-prepared grab-and-go meals, according to Tammy Gonzales, senior marketing manager, deli and ...prepared entrees at Wayzata, Minn.-based Cargill. According to Golub, the chain has had great success with special limited-time offers and is continually "looking for the next opportunity to meet the moment for our customers." ...61% of consumers are interested in Latin cuisine for the variety of flavors its recipes inspire.
Focus on Digestibility Lactose-free milk is one segment experiencing a surge among health-conscious consumers, due to its lower calorie content and easier digestibility, according to research from ...Future Market Insights. According to Bailey, availability of A2 milk products is good news for the millions of consumers who have trouble digesting milk. Among smaller companies, Austin, Texas-based frozen treat brand GoodPop has expanded into the beverage aisle with Mini Cans, lunchbox-size 7.5-ounce drinks made with real fruit juice and sparkling water, but no added sugars, sweeteners or flavors.
Prepared to the Max Sax, Barbara
Progressive Grocer,
09/2023, Letnik:
102, Številka:
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Trade Publication Article
Nielsen IQ data cited in FMI - The Food Industry Association's most recent "Power of Foodservice" report shows that deli prepared food dollar and unit sales grew in 2022, along with purchase ...frequency among heavy buyers. According to Hill, since prepared food and catering items are typically some of the most profitable items sold in-store, retailers have an opportunity to expand their offering for customers, grow loyalty and enhance their businesses overall. The Kroger Co., based in Cincinnati, has become the biggest sushi retailer in the United States, according to a recent report in The Wall Street Journal, while its Home Chef roasted chicken products are "wildly popular" with consumers, since these products "offer convenient and healthy options at affordable prices," says Teresa Sabatino, director of customer marketing at Home Chef.
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