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OBJECTIVE CONCLUSIONS Characterizing Mouse Mammary Epithelial Cell Response to Activin and TGFβ Natalia Trikoz, Varun Sheel, Amy L Roberts, Brittany Paquette, Karen A Dunphy University of Massachusetts Amherst INTRODUCTION RESULTS Cytokines Influence Mammary Gland Development and Tumorigenesis PRELIMINARY CDBGeo DATA METHODS TGFB induces persistent EMT in CDBGeo Cells ABSTRACT Epithelial to Mesenchymal Transition (EMT) Epithelial Cells Mesenchymal Cells Cells treated with cytokine 12 days prior to plating Cells treated with cytokine 15 days prior to plating Treatment of Cells In Vitro with Cytokine Mammosphere Assay qPCR: Gene Expression Amplification Curve qPCR Results qPCR Results Mammosphere Assay Mammosphere Assay Mammosphere: sphere of cells that originates from a presumed mammary stem cell that can survive and proliferate under non-adherent conditions Image Source: http://commons.wikimedia.org/wiki/File:PCR_with_SYBR_green.jpg Characterize responses to TGFβ and Activin in two other mouse mammary cell lines •TM9 – mildly tumorigenic •TM2H – highly tumorigenic Both cells lines are p53 deficient and are preneoplastic TM2H cells respond to TGFB, but not Activin. However effects, unlike CDBGeo cells, are transient, not persistent. 1)Phenotype • Mesenchymal morphology during treatment (d15), but return to epithelial morphology after withdrawal at cytokine (d21) 2) Gene Expression • Increased expression of Snail, but not ZEB2 • Decreased expression of MTAC2d (d12) is restored after withdrawal of cytokine (d21) 3) Mammospheres • No difference in primary mammosphere number of mammosphere forming efficiency • But TGFB-treated cells grow larger mammospheres with more cells • TGFB treated cells have increased self-renewal capability Cells treated with cytokine 12 days prior to plating Cells treated with cytokine 28 days prior to plating Transforming growth factor beta (TGFβ) and activin are cytokines that utilize common signaling pathways, via smad2/3 and smad4, to mediate tumor suppression by affecting cell cycle arrest and apoptosis. However, TGFβ and activin each have specific receptors allowing them the capability of divergent signaling. Differences in temporal expression patterns suggest that each cytokine has specific roles in mammary gland development. Activin is expressed during pregnancy and lactation and is required for branching and lactogenesis, implying a role in mammary gland maturation. In contrast, TGFβ is expressed during involution during mammary gland regression and functions to re-organize the mammary epithelial content to the non-lactating state. Previously, the Jerry/Dunphy Lab found that TGFβ (5ng/ml) and Activin (50ng/ml) do share common signaling pathways allowing both cytokines to restrict the growth of a transplantable mouse mammary epithelial cell line (CDβGeo). However, extended exposure to TGFβ causes epithelial to mesenchymal transition (EMT) with persistent activation of EMT genes including up- regulation of up-regulation of SNAIL and ZEB2, and down-regulation of MTAC2d. The TGFβ-exposed cells are also more tumorigenic. Activin-treatment does not induce EMT. In contrast, Activin-treated cells have increased expression of MTAC2d relative to controls. More importantly, Activin treatment decreased mammosphere forming efficiency and outgrowth potential in transplants indicating that progenitor potential was decreased (data not shown). Our objective was to characterize responses to TGFβ and Activin in two mouse mammary cell lines. Both cell lines are p53 deficient, preneoplastic and some will proceed to form tumors in transplants The TM9 cell line is mildly tumorigenic, while the TM2H cell line is highly tumorigenic.. Both cell lines were compared for growth rate and morphology, but only the TM2H line was assessed for response to cytokine. The TM2H cells are unresponsive to Activin. In contrast, TGFβ treatment inhibited cell growth and induced EMT in the TM2H cells. Cells lost epithelial (cobblestone) morphology and acquired a mesenchymal (spindle-like) morphology. During the treatment period (days 1-15) the TGFβ- treated cells demonstrated gene expression change indicative of EMT including up-regulation of Snail and down-regulation of MTAC2d, although Zeb2 was unchanged. However, when cytokine was withdrawn, the TM2H cells returned to their epithelial state. There was no difference in primary mammosphere forming capability between either cytokine and the controls. However, it was noticed that the TGFβ-treated cells generated significantly larger mammospheres. Dissociated, the TGFβ mammospheres had a significantly greater number of cells and they also had greater self-renewal capability, indicative of progenitor cells. We conclude that the TM2H cells undergo transient EMT in response to TGFβ, unlike the CDβGeo cells which undergo persistent EMT. Monolayer Day 14 (cytokine treatment) Day 21 (no cytokine treatment) Mammospheres TM9 TM2H Cell Line Characteristics • TGFB is a tumor suppressor and tumor promoter • Activin is a tumor suppressor , but it’s role in tumor progression is unknown
